In the revival of herbal age, aromatic crops are being commercially cultivated in order to fetch the great demand of essential oils used by food, pharmaceutical flavour, perfumery and cosmetics industries. Two isolates of plant growth promoting rhizobacteria (PGPR) which were isolated from the rhizosphere soil of Pyrethrum (Chrysanthemum cineraefolium) designated as MA-2 and MA-4, and identified as Bacillus subtilis and Pseudomonas fluorescence on the basis of cultural as well as biochemical testing. They gave excellent result on the productivity of Pelargonium graveolens, increased herb yield over control by 9 and 27.6% respectively.
Plant growth promoting rhizobacteria (PGPR) are originally defined as root- colonizing bacteria i.e. Bacillus subtilis and Pseudomonas fluorescence that cause either plant growth promotion or biological control of plant diseases (1). The potential to use PGPR in integrated strategies to reduce N and P fertilizers offers an appealing research area for those scientists engaged in growth promotion studies in dependable of biological control. As with attempts to employ PGPR for biological control, practical use of growth promoting PGPR will be aided by clear elucidation of mechanisms for growth promotion. There are several reports that PGPR's have promoted the growth of reproductive parameters of plants ranging from cereals, pulses, ornamentals, medicinal and aromatic plants, vegetable crops, and even tree species.
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Treatment with PGPR has increased the germination percentage, seedling vigor, emergence, plant stand, root growth, shoot growth, total biomass of the plants, seed weight, early flowering, increased grain, fodder, fruit yields etc. (2,3). The exact mechanism involved in growth promotion when agronomic crops are inoculated with rhizobacteria include, increase in the nitrogen fixation, the production of auxin, gibberellins, cytokinin, ethylene, the solubilization of phosphorus and oxidation of sulfur, increase in nitrate availability, the extracellular production of antibiotics, lytic enzymes, hydrocyanic acid, increases in root permeability (4). ACC (1-aminocyclopropane-1-carboxylate) deaminase activity, siderophore production, enhancing biological nitrogen fixation and enhancement in the uptake of essential plant nutrients could be the best possible explanations. It has been widely reported in numerous microbial species of gram negative bacteria (5). It is extensively studied in numerous species of plant growth promoting bacteria like Bacillus (6) and Pseudomonas (7).
Furthermore, the plants grow faster and greener with longer roots and shoots than the untreated plants. It has been established that fluorescent Pseudomonas enhance plant growth in several ways viz., producing plant growth regulators, such as gibberellins, cytokinins and indole acetic acid, which can either directly or indirectly modulate the plant growth and development (8,9).
Geranium Plant (Pelargonium graveolens L'Herit)
Geranium is an erect, much-branched shrub, that can reach a height of up to 1,3 m and a spread of 1 m. The hairy stems are herbaceous when young, becoming woody with age. The deeply incised leaves are velvety and soft to the touch due to the presence of numerous glandular hairs. The leaves are strongly rose-scented. The showy white to pinkish flowers is borne in an umbel-like inflorescence and is present from late winter to summer (August-January).
This plant is confined to two separate areas in Southern Africa, one in Limpopo Province, where it receives summer rain, and the other in the south-eastern part of the Western Cape, where it receives rain throughout the year. In both these regions, the summer is hot and the winter is mild, and Pelargonium graveolens is found growing on the mountains, in sheltered positions such as kloofs, usually in relatively moist habitats. Pelargonium graveolens has also been recorded in Zimbabwe and Mozambique. Pelargonium graveolens oil is used extensively in high class of perfumes, soaps and cosmetics because of its pronounced and lasting rose like odour due to rhodinal content. (10). The oil of Pelargonium graveolens is also used in aromatherapy.
Materials and Methods
Isolation of Rhizobacteria
The soil samples were randomly collected from rhizospheric soil of Pyrethrum (Chrysanthemum cineraefolium) at Central Institute of Medicinal and Aromatic Plant (CIMAP), Lucknow, India, and dried at room temperature for 24 hours. 1 gram of soil was dissolved in 10 ml of sterilized water in a test tube, vortexed at high speed and serially diluted to 1:10, 1:100, 1:1000 and 1:10000. An amount of 500 µL of each dilution was separately spread on petri plates containing nutrient agar and King's B medium (11). Three replicates were maintained for each sample. The plates were then sealed with parafilm, incubated at 30±2°C and growth was examined after 24-72 hours. Each of the colonies produced in petridishes were transferred in to fresh nutrient agar slant and cultures were stored at -20°C in refrigerator (12).
Biochemical characterization of rhizobacteria
Always on Time
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Selected isolates of Bacillus (MA-2) and Pseudomonas (MA-4) were biochemically characterized by Gram's reaction, carbohydrate fermentation, oxidase test, O-F test, H2S production, IMViC tests, NO2 reduction, starch hydrolysis, phosphate solublization test (TCP) and gelatin hydrolysis as per the standard methods (13,14).
Production of Indole acetic acid
Indole acetic acid (IAA) production was detected as described by Brick et al., (15). Bacterial cultures were grown for 48h (Bacillus and Pseudomonas) on their respective media at 36±2°C. Fully grown cultures were centrifuged at 3000 rpm for 30 min. The supernatant (2ml) was mixed with two drops of orthosporic acid and 4ml of the Salkowski reagent (50ml, 35% of perchloric acid, 1 ml 0.5M FeCl3 solution). Development of pink colour indicates IAA production.
Production of Ammonia
Bacterial isolates were tested for the production of ammonia in peptone water. Freshly grown cultures were inoculated in 10 ml peptone water in each tube and incubated for 48-72 h at 36±2°C. Nessler's reagent (0.5 ml) was added in each tube. Development of brown yellow colour was a positive test for ammonia production production (13).
Siderophore production was detected by the universal method of Schwyn and Neilands (16) using blue agar plates containing the dye chrom azurol S (CAS). Orange halos around the colonies on blue were indicative for siderophore production.
Application of rhizobacteria in Pelargonium graveolens
Single pure isolated colony of selected rhizobacteria was multiplied in nutrient broth medium by incubation for 4-5 days over rotatory shaker at 110 rpm. The bacterial culture was centrifuged at 10,000 rpm for 10 min. The pellet was collected and mixed in 0.01 MgSO4 with the help of magnetic stirrer. An amount of 10-ml bacterial culture was mixed in 100 g of vermicompost, which was applied around the root zone of Pelargonium graveolens L'Hérit. In control only 100g vermicompost was added. The effect of PGPR on the growth of Pelargonium graveolens was recorded after three months of inoculation.
Results and Discussion
In present study strain MA-2 and MA-4, isolated from Crysanthemum cineraefolium rhizosphere and also release inorganic phosphate in tri calcium phosphate medium, which were identified as Bacillus subtilis and Pseudomonas fluorescence respectively on the basis of cultural as well as biochemical testing (Table 1 and 2).
Table 1. Morphological, cultural and biochemical characteristic of rhizobacteria
Isolate No. MA-2
Isolate No. MA-4
Colony on nutrient agar
Irregular, undulate creamish dull, with ground glass appearance
Round with entire margin, creamish
Fluorescent on Kings B
Single and in chains
Growth at 30°C
Haemolysis in Blood agar
Clear zone (b-haemolysis)
Inverted tree +
Litmus milk reduction
Acid by reduction
A (-), G (-)
A (-), G (-)
A (+), G (-)
A (+), G (-)
A (-), G (-)
A (+), G (-)
Growth on PDA
A = Acid, G = Gas.
Similarly 44 bacterial isolates from the rhizosphere of tomato were screened for their plant growth promoting activities (17) are able to solubilize sparingly soluble phosphate, usually by releasing chelating organic acids (18). Our strain MA-4 produces fluorescent pigment on King's B agar medium, as reported for production of fluorescent pigment by Pseudomonas fluorescence (11).
Table 2. Plant growth promoting characteristics of rhizobacterial isolates
Isolate No. MA-2
Isolate No. MA-4
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Phosphate solubilizing test (TCP)
In our study both plant growth promoting bacterial isolates isolated from Chrysanthemum cineraefolium of Asteraceae and used against the member of Geraniaceae. While 107 rhizobacterial isolates, obtained from the rhizosphere of Eucalyptus spp. belongs to family Myrtaceae (19). Some plant growth-promoting rhizobacteria such as Bacillus subtilis RC11 were efficient in phosphate solubilization and indole acetic acid (IAA) production and significantly increased growth of wheat and spinach (20).
Therefore, in present study plant growth promoting rhizobacteria Bacillus subtilis strain MA-2 and Pseudomonas fluorescence strain MA-4 were efficient in phosphate solubilization and indole acetic acid (IAA) (Fig. 1) production and significantly increased biomass 9% and 27.6% respectively of medicinal and aromatic plant such as Geranium.
Figure 1. Phosphate solublization test of Bacillus subtilis (MA-2) and Pseudomonas flurescence (MA-4) on Tri calcium phosphate medium
Increased in biomass production leads to the essential oil yield (Table 3) (Fig. 2). The effect of some bacteria isolates on root formation, root length and dry matter content of roots of mint (Mentha piperita L.). Mint and Agrebacterium rubi (strain A16), Burkholderia gladii (strain BA7), Peseudomonas putidea (strain BA8), Bacillus subtilus (strain OSU142) Bacillus megatorium (strain M3) were used as rooting agent, respectively (21).
Table 3. Influence of PGPR's (Bacillus subtilis and Pseudomonas fluorescence), Isolate No. MA-2 and MA-4 on the productivity of Geranium (Pelargonium graveolens) in pots (unsterile soil)
Average plant height
Average no. of branches
Average herb yield fresh
wt. in (g)
%increase average herb yield fresh weight over control
According to Kohler et al., (22) inoculation with B. subtilis increased significantly the urease, protease and phosphatase activities of the rhizosphere soil of the lettuce plants and also increased foliar P and K contents. The mechanisms action of PGPR, namely, induced systemic resistance (ISR) and induced systemic tolerance (IST) were elaborated (23).
Figure 2. Effect of PGPR on the growth of Pelargonium graveolens (A) Bacillus subtilis
Fcalculated value of Pelargonium graveolens L'Hérit plant height due to treatment is 1.497 where as the F table value at 5% probability level is 4.10, treatment is statistically non significant because the calculated value of treatment is lower than the value of F(5%) table value.
Fcal £ F(5%) (Non significant)
Fcalculated value of Pelargonium graveolens L'Hérit herb yield fresh wt. Due to treatment is 11.39 where as the Ftable value at 5% probability level is 6.94 treatment is statistically significant because the calculated value of treatment is higher than the value of F(5%) table value.
Fcal ³ F(5%) (Significant)
Research in last decade has opened up new horizons for the inoculation industry. Agriculture in developed countries is definitely the major promoter of microbial inoculants that are 'environmentally friendly'. In recent years fluorescent pseudomonads have drawn attention world wide due to production of secondary metabolites such as siderophores, antibiotics, volatile compounds, enzymes and phytohormones. Strains of Bacillus subtilis and Pseudomonas fluorescence, gave effective result in growth promotion in medicinal and aromatics plant such as Geranium (Pelargonium graveolens), therefore called plant growth-promoting rhizobacteria. Isolate MA-4 (Pseudomonas fluorescence) gave better result on the productivity of Geranium in comparison to MA-2 (Bacillus subtilis). They also possessed biological control activities. Thus, they could be further exploited for commercial scale up.