Response of single and co-inoculation of plant growth promoting rhizobacteria on growth , flowering and nutrient content of chrysanthemum

A pot experiment was conducted in the screen-house of Department of Horticulture, College of Agriculture, CCS Haryana Agricultural University, Hisar during the two successive seasons of 2011-12 and 2012-13 to investigate the potential effect of different strains of Bacillus (BS1SYB101, BS2SB155 and BS3SB127), Pseudomonas (PS1WPS73, PS2CPA152 and PS3-P20) and their combination on growth, flowering and nutrient content of chrysanthemum. Strains of Bacillus and Pseudomonas significantly influenced the observed parameters of chrysanthemum. Maximum plant height and number of branches per plant were recorded in plants inoculated with PS2 strain of Pseudomonas (CPA152) and BS3 strain of Bacillus (SB127) in both the years. The minimum number of days taken to bud initiation, days for first flowering from bud initiation and days taken for 50% flowering were recorded in plants inoculated with PS2 strain of Pseudomonas (CPA152) and BS3 strain of Bacillus (SB127). The maximum flower size was noticed with PS3 strain of Pseudomonas (P20) in the first year whereas, in second year, the response of Pseudomonas strains was found non-significant. Among Bacillus strains, the plants inoculated with BS3 (SB127) recorded maximum flower size. Maximum flower yield/plant was recorded in plants inoculated with PS2 strains of Pseudomonas (CPA152) and BS3 strains of Bacillus (SB127). The N content of plant was recorded maximum with PS2 (CPA152) and PS3 (P20) strains of Pseudomonas and BS3 (SB127) strains of Bacillus, whereas, P content of plant was noticed maximum with PS3 strains of Pseudomonas (P20) in the first year, while it was found non-significant in second. The response of Pseudomonas strains to K content of plant was found non-significant in both years, while K content of plant was influenced significantly by Bacillus strains.


INTRODUCTION
Chrysanthemum (Chrysanthemum morifolium Ramat.) which occupies a prominent place in ornamental horticulture is one of the commercially exploited flower crops.It is mainly grown for cut and loose flowers for garland making, general decoration, hair adornments and religious function.Increased flower production, quality of flowers and perfection in the form of plants are the important objectives to be reckoned in commercial flower production.Though the quality of flowers is primarily a varietal trait, it is greatly influenced by climatic, geographical and nutritional factors among which nutrition play major role (Laishram et al., 2013).At present, these nutrients are supplied through chemical fertilizers.The use of chemical fertilizers has resulted not only in the deterioration of soil health but also has led to some major environmental problems, such as soil and water pollution and other health related problems, besides increasing the input cost for crop production especially on the marginal farmers.
Plant growth-promoting rhizobacteria (PGPR) are naturally occurring soil bacteria that aggressively colonize plant roots and benefit plants by providing growth promotion (Saharan and Nehra, 2011).They are a heterogeneous group of bacteria that can be found in the rhizosphere, at root surfaces and in association with roots, enhancing the growth of the plant either directly and/or indirectly (Glick, 1995).Inoculation of crop plants with certain strains of PGPR at an early stage of development improves biomass production through direct effects on root and shoots growth.They not only promote plant growth but also help in sustainable agricultural development and protecting the environment (Das et al., 2013).It is well established that only 1 to 2% of bacteria promote plant growth in the rhizosphere (Antoun and Kloepper, 2001).The mechanisms by which PGPR promote plant growth are not fully understood, but it is believed that the plant growth promoting rhizobacteria enhance plant growth and yield either by direct or indirect mechanisms (Glick, 1995).The direct promotion of plant growth by PGPR entails either providing the plant with a compound that is synthesized by the bacterium, for example plant growth regulators, or facilitating the uptake of certain nutrients from the environment (Glick, 1995).The indirect promotion of plant growth occurs when PGPR lessen or prevent the deleterious effects of one or more phytopathogenic organisms.This can happen by producing antagonistic substances or by inducing resistance to pathogens (Glick, 1995).A particular PGPR may affect plant growth and development by using any one, or more, of these mechanisms.PGPR, as biocontrol agents, can act through various mechanisms, regardless of their role in direct growth promotion, such as by known production of auxin phytohormone (Patten and Glick, 2002), decrease of plant ethylene levels (Glick et al., 2007) or nitrogen fixing associated with roots (Dobereiner, 1992).PGPR and their interactions with plants are exploited commercially and hold great promise for sustainable agriculture.Thus the aim of this study was to determine the effect of plant growth promoting rhizobacteria on growth, flowering and nutrient content of chrysanthemum.

MATERIALS AND METHODS
The present experiment was carried out in the screen-house of the Department of Horticulture, College of Agriculture, CCS Haryana Agricultural University, Hisar, India during the year 2011-2012 and 2012-2013.Hisar is situated at 29° 10 East longitude with an elevation of 215.2 m above mean sea level.The tract falls in the semi-arid subtropical region having the characteristic extremes of weather conditions with hot dry winds during summers and severe cold in winters.For experimental purpose, soil was collected from pure sand dune near to Hisar and mixed thoroughly.Each pot was lined with polythene sheet and filled with 5 kg of soil.The experimental soil was sandy in texture having 0.19% organic carbon, 47.5 ppm available nitrogen, 5 ppm available phosphorus and 51 ppm available potassium.One month old rooted cuttings of Chrysanthemum morifolium Ramat.cv.'Dolly Orange' having almost equal size (5-7 cm) and vigour were transplanted in the centre of pot in the month of September.Soil was firmly pressed around the plant and light watering was done immediately.The biofertilizers used in the experiment were procured from the Department of Microbiology, Chaudhary Charan Singh Haryana Agricultural University, Hisar, India during both the years of investigation.Three strains of Bacillus (BS1-SYB101, BS2-SB155 and BS3-SB127), three strains of Pseudomonas (PS1-WPS73, PS2-CPA152 and PS3-P20) and their combination were applied in three replications having CRD experimental design.In addition to the above treatments recommended dose of fertilizers that is NPK 30, 20, 20 g/m 2 or 150, 100, 100 ppm were also applied.Nitrogen, phosphorus and potassium were applied through urea (46% N), single super phosphate (16% P2O5) and murate of potash (60% K2O), respectively.Half dose of nitrogen and full dose of phosphorous and potash were applied as a basal dose just before planting of rooted cuttings, while the remaining half of the nitrogen was applied after 30 days of planting by top dressing method.The culture of Bacillus and Pseudomonas strains were grown in LB broth media for three days.About 20 ml suspension was applied to rhizosphere of the plant after 6 days of plantation as per treatments.Data on various growth, yield and quality parameters viz., plant height, number of branches per plant, number of days taken for bud initiation, number of days taken for first flowering from bud initiation, number of days taken for 50% flowering, size of flower (cm), flower (capitulum) yield per plant (g), and nutrient content in chrysanthemum plant (%) were recorded and average data were analyzed statistically as per method suggested by Panse and Sukhatme (1978).Estimation of nitrogen was done by Nessler's reagent method as per standard procedure (Jackson, 1967).Phosphorus content in plant sample was determined by 'Vanado-Molybdate method' (Jackson, 1967).The intensity of yellow colour was read at 430 nm in Elico spectrometer model CL-24.Potassium content of plant tissue was determined by flame photometer method.Readings were taken in Elico flame photometer; model C-140 after digesting the samples with triacid mixture (Jackson, 1967).

Plant height (cm)
The data recorded on the response of different strains of biofertilizers and their interactions on plant height are presented in Table 1.It is apparent from the data that plant height was significantly influenced by different strains of Pseudomonas (Figure 1) and the maximum plant *Corresponding author.E-mail: anopflori.25@gmail.com.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License It is inferred from the data that the effect of Bacillus strains was also found to be significant for plant height (Figure 2).The maximum plant height (27.69 and 26.99 cm) was observed with the application of BS 3 treatment in the year 2011-2012 and 2012-2013, respectively, but in the year 2012-2013, it was at par with BS 2 application with the value of 26.88 cm.The minimum plant height (23.53 and 24.12 cm) was recorded in control during both the years, respectively.Among the interactions of Bacillus and Pseudomonas strains, the maximum plant height (31.80 cm) was recorded with the inoculation of BS 3 PS 3 , which was at par with inoculation of BS 2 PS 2 (30.10 cm) in the year 2011-2012, whereas, in the year 2012-2013, the tallest plant (29.73 cm) was recorded with application of BS 2 PS 2 , which was at par with BS 3 PS 3 (28.10cm), BS 3 PS 2 (27.97 cm), BS 3 PS 0 (27.80 cm), BS 2 PS 3 (27.50cm) and BS 0 PS 2 (27.47 cm) treatments.The smallest plant (21.13 and 19.21 cm) was observed in control during both the years, respectively.Indole acetic acid (IAA) is one of the naturally occurring auxins with broad physiological effects of plants.Many rhizosphere bacteria including Bacillus, Pseudomonas, Azotobacter, Azospirillum, etc. were found to have the ability to produce IAA or related auxins (Salma et al., 2013).Auxins have been implicated in initiation of lateral and adventitious roots, in stimulation of cell division and elongation of stems and roots (Malamy and Benefy, 1997).Increased of plant height with the application of Bacillus (SYB101) and Pseudomonas (CPS63) strain in gladiolus was also reported by Singh   (2009).Dua and Sindhu (2012) reported that in wheat single inoculation of Pseudomonas strain WPS3 or WPS90 resulted in increased plant growth as compared to control.Such a positive effect was in line with the findings of Prasad et al. (2012) in chrysanthemum and Jayamma et al. (2008) in jasmine.

Number of branches per plant
The data regarding number of branches per plant are given in Table 2, which reveal that the number of branches per plant was influenced significantly by different strains of biofertilizers.In the year 2011-2012, the maximum number of branches per plant (7.17) was observed with the inoculation of PS 2 treatment, which remained at par with PS 3 (6.75),whereas, in the year 2012-2013, it was observed maximum with PS 3 (7.17),which remained at par with PS 2 (7.00).The minimum number of branches per plant (5.75 and 5.92 cm) was observed with control during both the years, respectively.It is inferred from the data that among the Bacillus strains, the number of branches per plant was noted maximum with application of BS 3 strain (7.17 and 7.33) in both the years, respectively, which was at par with the treatment of BS 2 (7.00) in the year 2012-13.The minimum number of branches per plant (6.00 and 6.08) was found with control during both the years, respectively.
The interaction effects of different strains of Bacillus and Pseudomonas on number of branches per plant was found significant in first year, whereas, it was nonsignificant in second year.During the year 2011-2012, BS 3 PS 3 and BS 1 PS 3 treatment combination recorded the maximum number of branches per plant (7.67), which was at par with BS 2 PS 2 and BS 3 PS 2 (7.33) and BS 3 PS 1

Number of days taken for bud initiation
The data regarding number of days taken for bud initiation are presented in Table 3.It is apparent from the data that number of days taken for bud initiation was significantly reduced by the inoculation of Pseudomonas strains.The minimum number of days taken for bud initiation (64.92 and 65.25 days) was recorded with PS 2 inoculation, which was at par with PS 3 (65.58and 65.50 days) in the year 2011-2012 and 2012-2013, respectively.The maximum number of days taken for bud initiation (72.17 and 71.92 days) was observed with control during both the years, respectively.The effect of Bacillus strains was also found to be significant with respect to number of days taken for bud initiation.The minimum number of days taken for bud initiation (69.67 and 65.33 days) was recorded with the inoculation of BS 3 treatment in the year 2011-2012 and 2012-2013, respectively, but in first year, it was at par with BS 1 (71.00 days) and BS 2 (72.00 days) treatments.The maximum number of days taken for bud initiation (76.00 and 70.08 days) was found in control during both the years, respectively.
The interaction between Bacillus and Pseudomonas strains was found to be significant during both the years.In first year, the minimum number of days taken for bud initiation (62.00 days) was recorded with inoculation of BS 3 PS 2 , which was at par with treatment of BS 0 PS 2 , BS 1 PS 3 , and BS 3 PS 3 (63.33days), BS 3 PS 1 (64.00 days) and BS 2 PS 2 (64.67 days).In second year, the minimum number of days taken for bud initiation (61.67 days) was recorded with inoculation of BS 1 PS 3 , which was at par with BS 3 PS 2 (62.00 days), BS 2 PS 2 (63.33 days), BS 3 PS 3 (64.00days), and BS 0 PS 2 (64.67 days) treatment combinations.The maximum number of days taken for bud initiation (76.00 and 74.33 days) was found with control during both the years, respectively.The earliness of bud initiation in biofertilizers inoculated plants may be ascribed to easy uptake of nutrients and simultaneous transport of growth promoting substances like cytokinins to the axillary buds, resulting in breakage of apical dominance (Jayamma et al., 2008).The present results are in confirmation with the findings of Singh (2009) who reported that combined application of Bacillus (SYB101) and Pseudomonas (CPS63) strains caused early spike initiation in gladiolus.The results are also in line with the findings of Salma et al. (2013) and Pandey et al. (2013) in gladiolus.

Number of days taken for first flowering from bud initiation
A close examination of data shown in Table 4 indicates that number of days taken for first flowering from bud initiation was significantly influenced due to different strains of Pseudomonas.The minimum number of days taken for first flowering from bud initiation (13.92 and 13.67 days) was recorded with PS 2 application and it was recorded maximum (18.17 and 17.48 days) in control during both the years, respectively.The response of Bacillus to number of days taken for flowering from bud initiation was also found significant in both the years.The minimum number of days taken for first flowering from bud initiation (14.17 and 13.50 days) was recorded with BS 3 application, whereas, the maximum number of days taken for flowering from bud initiation was seen in control (18.00 and 16.92 days) during both the years, respectively.Among the various combinations of Bacillus and Pseudomonas strains, the minimum number of days taken for first flowering from bud initiation (12.33 days) was recorded with BS 2 PS 2 and BS 3 PS 2 treatment combinations in first year, which was at par with BS 1 PS 3 and with BS 3 PS 3 (13.33days) treatments.In the second year, it was recorded minimum (11.67 days) with application of BS 3 PS 2 , which were at par with BS2PS2 (12.00 days) and BS3PS3 (12.33 days) treatment combinations.The maximum number of days taken for first flowering from bud initiation (20.67 and 19.67 days) was found with control during both the years, respectively.The probable reason for earlier flowering may be that the hormone secretion by Bacillus and Pseudomonas strains which enhance early bud initiation and flowering.Singh et al. (2010) reported that number of days taken for spike initiation reduced with the application of Bacillus and Pseudomonas strains in gladiolus.Barman et al. (2003) also observed that application of Bacillus reduced days required for flower opening in tuberose.

Number of days taken for 50% flowering
The data pertaining to response of different strains of Pseudomonas and Bacillus and their interaction on number of days taken for 50% flowering are presented in Table 5.The observation reveals that the minimum number of days taken to 50% flowering (97.92 and 97.17 days) was found in PS 2 inoculation and maximum The interaction effect of different strains of biofertilizers on number of days taken for 50% flowering was found to be non-significant in the first year, while it was significant in the second year, and the minimum number of days taken for 50% flowering (93.67 days) was recorded with inoculation of BS 3 PS 2 in the year 2012-2013, which was at par with BS 1 PS 2 (97.00 days) inoculation.The maximum number of days required for 50% flowering (112.67 and 113.33 days) was found in control in the year 2011-2012 and 2012-2013, respectively.The results of our experiment are in confirmation with the findings of Shivran et al. (2013) who reported that application of PGPR significantly decreased the number of days taken to 50% flowering fenugreek.Jayamma et al. (2008) also observed that plant receiving 50 per cent recommended dose of NPK fertilizers + biofertilizers took significantly lesser number of days for 50 per cent flowering as comparison to 100 per cent recommended dose of fertilizers.

Size of flower (cm)
The data on size of flowers as influenced by different strains of Bacillus and Pseudomonas and their interactions are furnished in Table 6.The biggest flower (4.55 cm) was recorded with the application of PS 3 treatment, which was at par with PS 2 (4.53 cm) in the year 2011-2012, whereas, in the year 2012-2013, the response of Pseudomonas strains was found to be nonsignificant.The smallest flower (4.09 and 4.08 cm) was recorded with control during both the years, respectively.Further, it is cleared from the data that application of BS 3 recorded the biggest flower (4.44 and 4.36 cm) in the year 2011-2012 and 2012-2013, respectively, which was at par with BS 2 application (4.42 cm) in first year, whereas, in second year, it was at par with BS 2 (4.31 cm) and BS 1 (4.30cm) treatments.The smallest flower (4.27 cm) was recorded in BS 1 inoculation during first year, whereas, in second year, it was recorded smallest (4.26 cm) with control.
The interaction effect of Bacillus and Pseudomonas strains on flower size was found to be significant in both the years.In the year 2011-2012, the biggest flower (4.72 cm) was recorded with treatment receiving BS 3 PS 3 strains, which was at par with BS 3 PS 2 (4.62 cm), while in second year, it was biggest with BS 1 PS 3 (4.54cm) treatment combination, which remained at par with BS 2 PS 2 (4.53 cm), BS 3 PS 2 (4.46 cm), BS 0 PS 2 (4.42 cm), BS 3 PS 3 (4.41cm), BS 0 PS 3 (4.40cm) and BS 1 PS 2 (4.38 cm) combinations.The smallest flower (3.91 and 3.87 cm) was recorded with control during both the years, respectively.The increased flower size might be due to the increased availability of nitrogen and phosphorus for flower development as a result of greater solubility and absorption of nutrients.Singh et al. (2010) also reported beneficial effect of different biofertilizers and their strains on floret diameter of gladiolus.These findings are in line to that of Pandey et al. (2013) who observed that application of Bacillus subtilis + vermicompost registered maximum diameter of floret in gladiolus.

Flower yield per plant (g)
The data pertaining to flower yield per plant have been presented in Table 7.The perusal of data elucidated significant differences amongst the different strains of  The interaction effects of Pseudomonas and Bacillus strains were found to be significant in both the years and the maximum flower yield per plant (69.07 g) was recorded with treatment receiving (Figure 3) BS 2 PS 2 in the year 2011-2012, whereas in the year 2012-2013, it was maximum with BS 3 PS 3 (70.93g),which was at par with BS 2 PS 2 (67.23 g) and BS 3 PS 2 (65.43g) treatment combinations.The minimum flower yield per plant (17.69 and 20.22 g) was recorded with control during both the years, respectively.A large body of evidence suggests that PGPR enhance the growth and crop yield, and contribute to the protection of plants against certain pathogens and pests (Kaushal et al., 2011;Dey et al., 2004;Kloepper et al., 2004;Herman et al., 2008 andMinorsky, 2008).

Nitrogen content (%) in plant (g)
The data on N content are presented in Table 8, which reveal that the different strains of Bacillus and Pseudomonas have significant effect on N content in plants.In the year 2011-2012, the maximum N content (3.02 %) was recorded with application of PS 2 , which was at par with PS 1 (2.82%) and PS 3 (2.82%) treatments, and the minimum N content (1.96%) was found with control.In the year 2012-13, the maximum N content (2.71%) was recorded with PS 3 , which was at par with PS 1 (2.48%) and PS 2 (2.36%) applications.Goel et al. (2002) reported that co-inoculation chickpea with Pseudomonas strains increase the nitrogen content in plants.
It is inferred from the table that the maximum N content (3.19%) was observed with treatment receiving BS 2 in first year, which remained at par with BS 3 (3.02%)and BS 1 (2.79%) treatments, whereas, in second year, the maximum N content (2.53%) was recorded with BS 3 treatment, which remained at par with BS 1 and BS 2 (2.51%).The minimum N content (1.69 and 2.07%) was found in control during both the years, respectively.The increase in concentration of nitrogen in the plant is due to the uptake of nitrogen by plants.The findings of Singh (2009) also revealed that nitrogen content in plant increased significantly with application of PGPR.The interaction effect was found to be non-significant in first year, whereas, it was significant in second year.The maximum N content (3.15%) was found with BS 1 PS 3 , which was at par with PS 0 BS 3 and BS 2 PS 1 (2.90%),BS 1 PS 2 (2.61%), BS 2 PS 3 (2.60%),BS 3 PS 3 (2.59%),BS 0 PS 3 and PS 0 BS 2 (2.51%) and BS 3 PS 2 (2.46%) treatment combinations, whereas, the minimum N content (0.92%) was noticed in control in the year 2012-2013.

Phosphorus content (%) in plant
The data pertaining to P content (%) are presented in Table 9.The perusal of data elucidates significant differences amongst the Pseudomonas strains for the year 2011-2012 but non-significant for the year 2012-2013.The maximum P content (0.36%) was recorded with PS 3 treatment, which was at par with PS 1 and PS 2 (0.32%), whereas, it was minimum (0.25%) in control during the year 2011-2012.The response of different strains of Bacillus on P content was found to be nonsignificant for both the year.The interaction effect of different strains of Bacillus and Pseudomonas was found to be significant and the maximum P content (0.43%) was recorded with the treatment combination of BS 1 PS 3 , which was at par with BS 0 PS 2 (0.37%) and BS 3 PS 1 (0.35%) in the year 2011-2012, whereas, it was nonsignificant in the second year.The minimum N content (0.20%) was recorded in control in the year 2011-2012.The beneficial role by biofertilizers may be ascribed as the cumulative activity of P-solubilization and phytohormones production.Barman et al. (2003) observed that NPK + FYM + PSB increased phosphorus content in leaves of tuberose from 0.04 to 0.06%.Similarly, Karishma et al. (2013) also reported that gerbera plants inoculated with mix culture of Glomus mosseae + Acaulospora laevis + Pseudomonas fluorescens showed maximum phosphorus content at lower concentration of superphosphate.

Potassium content (%) in plant
The data of K content in plants as influenced by different strains of Pseudomonas and Bacillus and their interactions are presented in Table 10, which reveals that the effect of Pseudomonas was found to be nonsignificant for both the years, whereas the effect of Bacillus strains on K content was found to be significant for both the years.The maximum K content (1.69 and 2.10%) was recorded with treatment BS 1 which was at par with BS 2 (1.51 and 1.78%) and BS 3 (1.57and 1.77%) for both the year, respectively.According to Kohler et al. (2007), inoculation with Bacillus subtilis increased significantly the urease, protease and phosphatase activities of the rhizosphere soil of the lettuce plants and increased foliar P and K contents.The interaction effect of Pseudomonas and Bacillus strains on K content was found to be non-significant during both the years of investigation.

Conclusion
The use of PGPR as inoculants biofertilizers is an efficient approach to replace chemical fertilizers and pesticides for sustainable flower cultivation.Among different strains of Bacillus and Pseudomonas, SB127 (BS 3 ) and CPA152 (PS 2 ) were found effective in increasing growth, flowering and yield parameters.The BS 2 PS 2 (SB155 + CPA152), BS 3 PS 2 (SB127 + CPA152) and BS 3 PS 3 (SB127 + P20) combinations showed best result with respect to growth, flowering and yield parameters of chrysanthemum.

Figure 2 .
Figure 2. Effect of different Bacillus strains on chrysanthemum.

Figure 3 .
Figure 3.Effect of different combinations of Bacillus and Pseudomonas strains on chrysanthemum.

Table 1 .
Response of single and co-inoculation of PGPR on plant height (cm) in chrysanthemum.

Table 2 .
Response of single and co-inoculation of PGPR on number of branches per plant in chrysanthemum.

Table 3 .
Response of single and co-inoculation of PGPR on number of days taken for bud initiation in chrysanthemum.
Jayamma et al. (2008)in number of branches per plant could be attributed to increased uptake of nutrients and increased activity of hormones like auxins, cytokinins, etc. in biofertilizers-inoculated plants.They also help in the absorption of relatively immobile nutrients such as P, Zn, Cu, Mn, Fe, etc. Increased number of primary and secondary branches per plant in fenugreek was also reported byShivran et al. (2013)with the application of different bioformulations.Similar findings have also been reported byPrasad et al. (2012)in Chrysanthemum indicum andJayamma et al. (2008)in jasmine.

Table 4 .
Response of single and co-inoculation of PGPR on number of days taken for flowering from bud initiation in chrysanthemum.

Table 5 .
Response of single and co-inoculation of PGPR on number of days taken for 50% flowering in chrysanthemum.

Table 6 .
Response of single and co-inoculation of PGPR on size of flower (cm) in chrysanthemum.

Table 7 .
Response of single and co-inoculation of PGPR on flower yield per plant (g) in chrysanthemum.

Table 8 .
Response of single and co-inoculation of PGPR on N content (%) in chrysanthemum plant.

Table 9 .
Response of single and co-inoculation of PGPR on P content (%) in chrysanthemum plant.

Table 10 .
Response of single and co-inoculation of PGPR on K content (%) in chrysanthemum plant.