International Journal of Agricultural Research

Year: 2006 | Volume: 1 | Issue: 6 | Page No.: 573-581
DOI: 10.3923/ijar.2006.573.581

Growth Responses of Helianthus annus to Plant Growth Promoting Rhizobacteria used as a Biofertilizers

Kamran Shaukat, Shazia Afrasayab and Shahida Hasnain

Abstract: Bacterial strains of Pseudomonas (Ps₁, Ps₂, Ps₃, Ps₄, Ps₅) Azotobacter (Ab₁, Ab₂, Ab₄, Ab₅, Ab₆) and Azospirillum (As₂, As₄, As₅, As₆, As₈) were used to inoculate the seeds of Helianthus annus var SF-187. Inoculated and non-inoculated seeds were germinated and grown under field conditions until at maturity. Two harvests were taken one after 7 days of germination and other at maturity i.e., after 4 months of sowing. All the bacterial inoculations provoked germination in Helianthus annus. Maximum increase was manifested by Ps₅ (223.63%). In general strains of Pseudomonas promoted plant growth more then Azotobacter and Azospirillum, plant height was enhanced by Ab₆ (68.66%) and number of leaves was increased by Ps₁ (63.63%) after 7 days of germination. However Ps₂ strain exhibited maximum enhancement in plant height (57.33%), while Ab₁ manifested maximum enhancement in number of leaves (49.01%) after 4 months of sowing. In case of yield parameters As₈ manifested maximum increase in number of flowers (128.57%), Ps₅ increased diameter of flowers (134.05%), Ab₁ weight of seeds (57.00%) and Ab₄ (30.35%) manifested enhancement in oil content when compared with control. Biochemical parameters were also increased with bacterial inoculations soluble protein content was enhanced by Ps₅ (321.55%), after 7 days of germination and Ab₂ (345.45%) after four months of sowing, auxin contents content was enhanced by, Ps₁ (1138%) and Ab₅ (845.50%), peroxidase activity was enhanced by Ps₅ (321.55%) and Ab₂ (345.45%) and acid phosphatase activity was increased by Ps₁ (97.13%) and As₈ (313.33%), after 7 days of germination and after four months of sowing when compared with that of control. These results demonstrate that these strains can be used for enhancing biomass as well as yield parameters.

Keywords: Azospirilum, Azotobacter, Biofertilizer and Pseudomonas

Introduction

Every increasing population of the world demands the increase in food production which intern depends upon the improved agricultural practices. Pakistan is an agriculture based country about 60% of our population is currently related to the agriculture. Agriculture contributed 24.1% in the year 01-02 (Federal Bureau of Statistics of Pakistan, 2000-02). Sunflower as an oil crop was introduced in Pakistan during early 1960s and its commercial cultivation began in 1965. Among the three NC oilseed crops, the sunflower has been found the most successful. Pakistan is the fourth biggest edible oil importer of the world, so it spends lot of money for edible oils. Overall Pakistan expends 6, 34,630 (million Rupees) during the year 2001-02. Out of this import expenses edible oil contributes 19,045 (million Rupees) in 2000-01 and was increased to 24,034 (million Rupees) during the year 2001-2002 (Federal Bureau of Statistics Pakistan 2000-02). So Pakistan will currently spend their huge amount on their edible oil import. Modern agriculture aims to increase crop yield to satisfy the needs from a growing population, but to use sustainable approaches that should include the substitution of chemical inputs by a more effective use of natural resources. Biological nitrogen (N₂) fits well in this model, as it is a more environmentally clean way to satisfying plant N needs. Now a day’s term PGPR is used for the bacteria’s, which enhanced the plant growth and productivity in one or more then one ways. The mechanism, which plant growth attributes to promotion, enhanced plant hormones concentration, (ii) asymbiotic nitrogen fixation, (iii) antagonizing against phytopathogenic microorganisms and (iv) solubilisation of mineral phosphates and other nutrients (Burd et al., 2000). Inoculation must, therefore be rhizosphere competent and survive in the soil to promote plant growth. Bacteria are very important for the physical and the chemical characteristics of soil and play important roles in soil quality and plant productivity (Hill et al., 2000). Acetobacter diazotoophicus, Herbaspirillum sereopedical, Azoarcus sp. and Azotobacter. Azospirillum are predominantly surface colonizing bacteria while others are endophytic diazotrophs (Steenhoudt and Vanderleyden, 2000). In the free-living diazotrophs Klebsiella pneumoniae and Azotobacter vinelandii, activation of expression of genes involved in nitrogen fixation by the enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo (Money et al., 2001). The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL, which bears significant homology to the histidine protein kinase transmitter domains (Money et al., 2001). In several diazotrophic species of Proteobacteria, P_II signal transduction proteins have been implicated in the regulation of nitrogen fixation in response to NH₄⁺ by several mechanisms. In Azotobacter vinelandii, expression of nifA, encoding the nif-specific activator, is constitutive and thus, regulation of NifA activity by the flavoprotein NifL appears to be the primary level of nitrogen control (Rudnick et al., 2002). According to Raza et al. (2001) in all lupin cultivars plant dry weight, growth and biomass production enhanced with rhizobial inoculation. PGPR Pseudomonas putinda KT2440 strain colonizes the Rhizosphere of a number of agronomically important plants at high population densities (Uregel et al., 2000; Turnbell et al., 2001). Yuming et al. ( 2003) three Bacillus strains, B. subtilis NEB4 and NEB5 and B. thuringiensis NEB17 enhanced soybean nodulation and growth in greenhouse and field experiments. Coinoculation with non-Bradyrhizobium endophytic bacteria provided the largest and most consistent increases in nodule number, nodule weight, shoot weight, root weight, total biomass, total nitrogen and grain yield. Dry and wet inoculation of alginate microbeads enhanced plant growth promotion in wheat and tomato seedling growing in unfertile soil and biodegraded within 15 days in moist soil (Bashan and Hernandez, 2002). Growth promoting bacteria induces systemic resistance in host plants (Zheng et al., 2000; Ramammorthy et al., 2000). Since many trials have used gnotobiotic or greenhouse conditions to show promotion, we examined weather the addition of indigenous monoculture inoculations of Pseudomonas, Azotobacter and Azospirillum used as Biofertilizer has effect on Helianthus annus var SF-187 in field. Inoculation effects on germination, growth and yield parameters were studied in field along with biochemical analysis (auxin content, protein content, peroxidase content acid phosphatase content and oil contents) and data was statistically analyzed.

Materials and Methods

The present study was conducted in the Botanical Garden, University of The Punjab, Quaid-e-Campus Lahore, Pakistan. The experimental site was located at longitude 74° East and latitude 31° north, which is at the altitude of 644.4 feet above sea level. The research was initiated in last week of December, 2001. Seeds of Helianthus annus were taken from Punjab Seed Corporation Lahore, which is certified from Federal Seed Certification and Registration Department Islamabad. Fifteen bacterial strains five each of Azotobacter, Ab₁, Ab₂, Ab₄, Ab₅ and Ab₆, Azospirillum. As₂, As₄, As₅, As₆ and As₈ Pseudomonas i.e., Ps₁, Ps₂, Ps₃, Ps₄ and Ps₅ isolated by Aziz (2000) were used in this study. Bacterial pellets obtained from fresh cultures (24 h incubation) of each strains and the cell density of these bacterial cultures were adjusted to 10⁸ cell mL^-1 with the help of 2-D spectrophotometer. Healthy seeds of Helianthus annus var were surface sterilized by soaking in 0.1% HgCl₂ solutions for 5 min with continuous shaking. Then seeds were left soaked in sterilized distilled water for about an hour. Sterilized seeds were then soaked in bacterial suspension with the help of sterilized forceps for about 15-20 min. For control treatment seeds were soaked in sterilized glass distilled water for same duration. Both inoculated and un-inoculated (control) seeds were then sowed in field. Germination was recorded daily for eight days and after that thinning was done in each plot. Two harvests were taken one after 7 days of germination and second at maturity i.e., after 4 months of sowing. Growth parameters such, plant height number of leaves with yield parameters were observed i.e., emergence of flowers, plant height, color of flowers and after that crop was harvested at maturity when pollination was completed and seed become mature i.e., after 4 months of sowing. For biochemical analysis at both harvests activity of peroxidase (David and Murray, 1965), acid phosphatase (Iqbal and Rafique, 1987), soluble protein content (Bhatti et al., 1993) and auxin content were measured by Mahadevan (1984). Data obtained was analyzed statistically following method of Steel and Torrie (1981). Mean, Standard error of the mean, least significant difference and correlation were calculated.

Results

Harvest I
Germination and Growth Parameters
In majority of cases bacterial inoculation provoked germination and 7.14% (Ps₂) to 110.74% (As₈), increases in germination of seeds were recorded (Table 1). Plant height or seedling lengths of Helianthus annus were enhanced significantly and it ranged from 15.85% (Ab₄) to (68.68%) Ab₆ (Table 1) when compare with that of control_. Bacterial strains Ps₂, Ab₅ and As₈ showed no effect on number of leaves per seedlings. Increases in this parameter ranged from 9.09% (Ps₃) to 63.63% (Ps₁). Whereas inoculation with Ab₁ (9.09%) and As₂ (9.09%) caused some reduction, relative to control, in this parameter (Table 1).

Harvest II
Growth and Yield Parameters
Plant height was increased significantly, with inoculation of bacterial strains, except for As₈ (6.11%), where decrease was recorded. The maximum increase in this parameter was observed with the inoculation of As₂ (64.74%) (Table 2). Among Pseudomonas strains 15.08% (Ps₄) to 54.38% (Ps₁) increases, in case of Azotobacter strains 6.19% (Ab₆)-48.48% (Ab₁), whereas in Azospirillum strains increases ranged from 2.416% (As₈) to 64.74% (As₂), relative to the non-inoculated control (Table 2). Bacterial inoculations increased number of leaves increase in this parameter, over control, ranged from 1.96-49.01%, however As₈ (21.56%) exhibited decreased in this parameter. Overall Azotobacter strains exhibited more stimulatory effects in this parameter, relative to Pseudomonas and Azospirillum strains. Number of flowers of Helianthus annus increased significantly, with the inoculation of bacterial strains, except As₂, which caused 28.57% reduction in this parameter, as compared to that of non-inoculated plants. The percentage increase in this parameter varied between 1.07 - 110.71%, with different strains, (Table 2). Generally Azospirillum strains manifested more increases then Pseudomonas and Azotobacter strains. The significant increase in diameter of flowers of Helianthus annus was recorded with all bacterial inoculations as compared to control. Increases in this parameter, relative to non-inoculated control, were 58.41-134.15%. The maximum increase was recorded with inoculation of As₅ (134.15%), relative to other bacterial inoculations (Table 2). With Pseudomonas strains percentage increases varies between 99.505% (Ps₁) to 130.71% (Ps₄), in case of Azotobacter strains increases were 58.24% (Ab₂) to 126.53% (Ab₆) and in Azospirillum strains increases ranged from 98.01% (As₂) to 134.14% (As₅), relative to non-inoculated control. All the bacterial inoculations increased weight of seeds in Helianthus annus plants (Table 2), when compared with control. Increase in this parameter ranged from 5.62 % (As₆) to 57.00% (Ab₁). In case of Pseudomonas strains maximum increase was exhibited by Ps₅ (44.85%), while Ps₁ (10.28%), showed minimum increase when compared with that of control. In case of Azotobacter strains increases in this parameter varies from 10.24% (Ab₅) to 57.00% (Ab₁) and in Azospirillum strains increases were 5.60% (As₆) to 34.11% (As₅), when compared with that of non-inoculated control. The oil contents of Helianthus annus seeds was increased with inoculation of most of bacterial strains, excluding Ps₁, Ab₅, As₂, As₄, As₆ and As₈, which caused reduction in this parameter, The maximum increase over non-inoculated control was recorded with inoculation of Ab₄ (30.35%) (Table 2). The increase in this parameter varied with different bacterial inoculations from 0.27% (Ps₄) to 18% (Ab₄). Azotobacter strains manifested more increases relative to that of Pseudomonas and Azospirillum strains Helianthus annus var SF-187.

Biochemical Analysis: (At Both Harvests)
Growth hormones are the substances produced in plants and these also affect the plant growth and yield, out of these distinct hormones, auxin appear to be a master hormone, exercising regulatory action over many different sorts of plant processes and probably over many of other plant hormones. Auxin content of Helianthus annus seedlings was enhanced significantly, over control, with the bacterial inoculations. Increase in auxin content ranged from 115.30% (As₈) to 1138% (Ps₁) after 7 days. However Pseudomonas strains showed relatively more increases in auxin content then Azospirillum and Azotobacter strains (Table 3). However after four months of sowing 36.36 (Ps₄)-854.35% (Ab₅) increases in auxin contents, over non-inoculated control, were recorded (Table 4). Pseudomonas strains Ps₂, Ps₅, Azotobacter strains Ab₁, Ab₄, AB₅ and Azospirillum strains As₅ and As₆ stimulated more than four folds increase in the auxin contents of sunflower plants (Table 4). Soluble protein contents were increased significantly over non-inoculated plants with the inoculation of many bacterial strains. after 7 days. However maximum increase was observed with the inoculation of Ps₅ (494.23%) (Table 3). The significant increase, in soluble protein contents of Helianthus annus was also recorded after four months of sowing, increases in this parameter, with bacterial inoculation ranged from 7.51% (Ab₂) to 41.69% (Ps₅) (Table 4).

Generally Pseudomonas and Azospirillum strains exhibited more stimulation in protein contents of Helianthus annus plants than Azotobacter strains. Enzymes are the biocatalysts, which play important roles in many biochemical reactions. Two enzymes acid phosphates and peroxidase were studied in this respect. Activity of peroxidase increased with bacterial inoculations and it ranged from 104.50% (As₆) to 362% (Ab₂) (Table 3) when compared with control after 7 days. Among Pseudomonas strains Ps₅ (321.50%) exhibited maximum enhancement, among Azotobacter strains Ab₅ (64%) manifested maximum increase whereas As₈ (104.56%) showed maximum increase in Azospirillum strains, when compared with control. however after four months of sowing peroxidases activity was increased significantly, relative to control, except for Ab₆, 18.93% (Ps₄) to 345.45% (Ab₂), increase was recorded after 4 months of sowing (Table 4). Acid phosphatase activity of inoculated seedlings increased significantly with the exception of As₅ which showed 15.35% reduction in this parameter. The increases varied between 2.716% (As₈) to 97.13% (Ps₁), when compared with control, after 7 days (Table 3). However after four months of sowing maximum increase over non-inoculated control was recorded with the inoculation of As₈ (313.33%) (Table 4).

Discussion

Bacteria are very important for the physical and the chemical characteristics of soil and play important roles in soil quality and plant productivity and play important roles in soil quality and plant productivity (Hill et al., 2000). In this study all the bacterial strains used to inoculate Helianthus annus var. SF-187 seedlings also proven to be efficient in plant growth promotion. All the strains used increased germination. In all cases bacterial inoculations significantly enhanced the plant height, number of leaves at both harvests. In case of Helianthus annus plant height was enhanced by Ab₆ (68.66%) and number of leaves was increased by Ps₁ (63.63%), after 7 days of sowing (Table 1). However Ps₂ strain exhibited maximum enhancement in plant height (57.33%), while Ab₁ manifested maximum enhancement in number of leaves (49.01%), after 4 months of sowing (Table 2). Improved seedlings growth with bacterial inoculation has also been reported by Hasnain and coworkers (Mehar et al., 2002). Rhizospheric bacteria Pseudomonas sp., Azospirillum sp. and Agrobacterium sp., increased plant growth and the nutrient uptake of maize, wheat and legumes (Bashan et al., 2004). With increase in plant height number, diameter of flowers and weight of seeds were also increased, As₈ manifested maximum increase in number of flowers (128.57%), (4.22), Ps₅ increased diameter of flowers (134.05%),(4.23), Ab₁ weight of 100 Seeds (57.00%), (4.24) and Ab₄ (30.35%), (4.25), manifested enhancement in oil content when compared to control (Table 2). This increase might be due to increase in availability of nutrients and nitrogen fixation deareasing pathogenecity and increased in protein contents. Burd et al. (2000) reported that plant growth promoting rhizobacteria might enhance plant height and productivity by synthesizing phytochromes, increasing the local availability of nutrients, facilitating the uptake of nutrients by the plants decreasing heavy metal toxicity in the plants antagonizing plant pathogens and inducing systemic resistance in the plants to pathogens. Acetobacter diazotoophicus, Herbaspirillum sereopedical, Azoarcus sp. and Azotobacter. Azospirillum are predominantly surface colonizing bacteria while others are endophytic diazotrophs (Steenhoudt and Vanderleyden, 2000). Fixation of atmospheric nitrogen (N) by free-living soil microorganisms is considered a minor source of bioavailable nitrogen compared to systems such as the Rhizobium-legume and Frankia-alder symbioses (Kennedy and Islam, 2001). In the free-living diazotrophs Klebsiella pneumoniae and Azotobacter vinelandii, activation of expression of genes involved in nitrogen fixation by the enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo (Money et al., 2001). The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL, which bears significant homology to the histidine protein kinase transmitter domains (Money et al., 2001). In several diazotrophic species of Proteobacteria, P_II signal transduction proteins have been implicated in the regulation of nitrogen fixation in response to NH₄⁺ by several mechanisms. In Azotobacter vinelandii, expression of nifA, encoding the nif-specific activator, is constitutive and thus, regulation of NifA activity by the flavoprotein NifL appears to be the primary level of nitrogen control (Rudnick et al., 2002). According to Raza et al. (2001) in all lupin cultivars plant dry weight, growth and biomass production enhanced with rhizobial inoculation. PGPR Pseudomonas putinda KT2440 strain colonizes the Rhizosphere of a number of agronomically important plants at high population densities (Uregel et al., 2000; Turnbell et al., 2001). Yuming et al. (2003) three Bacillus strains, B. subtilis NEB4 and NEB5 and B. thuringiensis NEB17 enhanced soybean nodulation and growth in greenhouse and field experiments. Growth promoting bacteria induces systemic resistance in host plants (Zheng et al., 2000; Ramammorthy et al., 2000). Endophytic nature of plant makes the PGPR to colonize and persist in intercellular spaces of epidermal cell (Ramammorthy et al., 2000). Bascones et al. (2000) reported that nitrogenase dependent hydrogen production is one of the major factors that influence the efficiency of symbiotic nitrogen fixation. Evidence of establishment of significant populations of these bacteria in the plant root surfaces in sprase, although many studies have demonstrated their occurrence in Rhizosphere soil surrounding the roots (Rammamooarthy et al., 2000). Bashan et al. (2000) reported that inoculation with various PGPB improved seed N, protein and P contents of Salicornia sp., which is an oilseed sp. In present study increase in yield of crop might be due to induced systemic resistance against pathogens. Raza et al. (2001) reported that analyses of plant dry weight showed that rhizobial inoculation positively influenced growth and biomass production for all lupin cultivars tested compared to with non-inoculated plants. The soluble protein contents, auxin contents, acid phosphatase and peroxidase activity were markedly increased with bacterial inoculations and this increase relates with the increase in growth and yield parameters of Helianthus annus. Soluble protein contents were also improved, with bacterial inoculation as compared to non-inoculation control, however in Helianthus annus Ps₅ (321.53%) manifested maximum increase after 7 days of germination (Table 3) and Ps₅ (41.62%) after four months of sowing as well (Table 4). PGPR improved seed N, protein and P contents of salicornia sp. when inoculated with PGPR (Bashan et al., 2000). Increase in N and protein contents of seeds 4-5 times with Azospirillum halopraeterens reported by Bashan et al. (2000). Growth hormones are the substances produced in plants and these also affect the plant growth and yield, out of these distinct hormones, auxin appear to be a master hormone, exercising regulatory action over many different sorts of plant processes and probably over many of other plant hormones. In Helianthus annus Ps₁ (1138%) exhibited maximum enhancement after 7 days of germination (Table 3) and Ab₅ (845.50%) after four months of sowing (Table 4). Enzymes are the biocatalysts, which play important roles in many biochemical reactions. Two enzymes acid phosphates and peroxidase were studied in this respect. Activity of enzymes, peroxidases was improved with inoculations of bacterial strains when compared with non-inoculated control, in Helianthus annus Ps₅ (321.55%) (Table 3) and Ab₂ (345.45%) (Table 4) exhibited maximum increase, Activity of enzyme acid phosphatase also improved with inoculation of bacterial strains. Ps₁ (97.13%) (Table 3) showed maximum increase after 7 days of germination. However after four months of sowing As₈ (313.33%) manifested maximum enhancement in Helianthus annus (Table 4). Bashan et al. (2000) reported that inoculation of oil seed halophytes Salicornia bigelovii with Azospirillum halopraeferens increased N and protein contents 4-5 times. Increase in protein content with bacterial inoculations is also reported by Afrasayab and Hasnain (2000 a, b). Two PGPR Bacillus pumilus and B. licheniformis produced high amounts of C1, - GAs, G_A1, GA₃, GA₄ and G₂₀ and biological data suggested that the bacterial media have the potential to elongate the stem and shoots of Alnus glutinosa (Manero et al., 2001). Bashan et al. (2000) reported that inoculation of oil seed halophytes Salicornia bigelovii with Azospirillum halopraeferens increased N and protein contents 4-5 times. Increase in protein content with bacterial inoculations is also reported by Afrasayab and Hasnain (2000 a,b).

Aon and Colaneri (2001) reported that enzymes have strong coorelation that enzymes activity play important role between physical, chemical and microbial soil properties, which ultimately enhanced plant growth. Two peroxidase and one Chitinase (35 k) isoforms have been induced in the PGPR treated plants inoculated with the rice sheath, blight pathogen Nandakumar (1998). It has been reported by Chen et al. (1999) that a novel acid phosphatase containing phosphotryrosyl phosphatase activity. Acid phosphatases from several bacterial species have been recognized as virulence factors that support intracellular survival by inhibiting the respiratory burst (Chhatwal et al.,1997; Reilly et al., 1996).

Conclusions

It is concluded from the above discussion that in Helianthus annus var SF-187 all the bacterial strains stimulated significantly growth and yield parameters at both harvests in field conditions, when compared with that of control. Increase in growth parameters (shoot length, root length and plant height) as well as yield parameters (Number and diameter of flowers, weight of seed, oil contents) at both stages was associated with increase in auxin and protein contents as well as peroxidase and acid phosphatase activities. Azotobacter and Azospirillum are non-symbiotic nitrogen fixer. It appears that these strains enhanced plant growth and yield parameters by applying nitrogen and synthesizing more auxin and proteins. Hence the strains used in the present study can be used as biofertilizer for the improvement of growth and yield parameters of commercially important cash crop i.e. Helianthus annus var. SF-197.

REFERENCES