pseudomonas aeruginosa strains and their role in plant growth promotion in medicinal plant

8/6/2019 Pseudomonas aeruginosa Strains and their Role in Plant Growth Promotion in Medicinal Plant

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Global Journal of Applied Agricultural Research.

Volume 1, Number 1 (2011), pp. 49-55

Research India Publications

http://www.ripublication.com/gjaar.htm

Pseudomonas Aeruginosa Strains and their Role in

Plant Growth Promotion in Medicinal Plant

Vishal Kumar Deshwal*, M.S. Devi, N. Bhajanka, J. Mistri,

A. Bose and N. Saini

Department of Microbiology, Doon (P.G.) Paramedical College,Dehradun-248001, Uttarakhand, India.

*Correspondence Author E-mail: [email protected]

Abstract

Total 20 Pseudomonas strains were isolated and characterized on the basis of

gram staining and various biochemicals tests. Further, these Pseudomonas

aeruginosa strains were selected for present study. These strains were screen

on the basis of plant growth activity like IAA, HCN, Siderophore and P-

Solubilization. All strains showed some plant growth activity but only fivePseudomonas aeruginosa strains i.e. MR-2, MR-5, MR-9, MR-15 and MR-18

produced all four Plant growth acivity like siderophore, HCN, IAA and P-

Solubilization. Pot experiment was carried out to analyse the plant growth

activity of these strain in medicinal plant. All bacterized Pseudomonas strains

produced more dry weight and plant height as compared to control treatment.

Pseudomonas aeruginosa MR-9 enhanced maximum plant dry weight, plant

height, nodule per plant, nodule fresh weight per plant by 184, 124, 139, 180%

respectively as compared to control.

Keywords: Pseudomonasaeruginosa, medicinal plant, PGPR.

IntroductionThe chemical agriculture affected the soil environment is well known. Biofertilizer

such as microbial inoculant which can promote plant growth and productivity have

internationally been accepted as an alternative source of chemical fertilizer. PGPR

represent a wide variety of soil bacteria which, when grown in association with a host

plant, result in stimulation of growth of their host [1]. Few strains from genera such as

Pseudomonas, Azospirillium, Azotobacter, Bacillus, Burkholderia, Enterobacter,

Rhizobium,Erwinia and Flavobacterium are well known PGPR [2, 3].


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50 Vishal Kumar Deshwal et al

Occurance of species of Pseudomonas in rhizosphere of various crop plants has

been mentioned by [4, 5]. Application of plant growth promoting rhizobacteria

increased the leaf number and area as well as number of annual shoot and its diameter

in apple [6]. PGPR inoculation significantly increased the root properties (length,

volume, mass) and shoot growth, the plant height (42-50%), leaf area (128-134%) [7].

Plant growth promoting rhizobacteria (PGPR) effectively colonized and enhanced

seed germination, shoot length, root length, fresh seedling weight, dry seedling weight

and nodule number in peanut plant (Arachishypogaea L.) [8].

Mucuna pruriens belongs to the family Fabaceae. It has some medicinal value and

it is also food feed crop. Seeds are astringent, laxative, anthelmentic, alexipharmic

and tonic [9], management of Parkinsons disease [10] and increase male sex

hormones -testosterone levels [11]. So the objective of this study was the evaluate

effect ofPseudomonas aeruginosa strains on plant dry weight, plant height, numberof nodule and nodule fresh weight of medicinal plant.

Materials and MethodsIsolation and purification ofPseudomonas stainsPseudomonas strains were isolated from rhizosphere of Mucuna plant. 1g of

rhizospheric soil dissolved in 9ml sterilized distilled water in test tube and mixed

well. Sample was diluted up to 105. 0.5ml sample of each tube was spread on separate

Kings B Agar plates. Plates were incubated at 37oC for 24hr. Fluorescent colonies on

agar medium were purified.

Characterization ofPseudomonas strains

One hundred isolates strains were characterized on the basis of gram staining and bio-

chemicals tests. Such tests were done according to Bergey's manual of Determinative

Bacteriology [12].

Screening of Plant growth promoting activity ofPseudomonas strains

Twenty Pseudomonasaeruginosa strains were screen and renamed as Pseudomonas

MR-1 to MR-20. These strains were screen on the basis of plant growth promoting

activity such as IAA, HCN, siderophore and P-solubilization.

Indole production testTryptophan, an essential amino acid, is oxidized by some bacteria by the enzyme

tryptophanase resulting in the formation of indole, pyruvic acid and ammonia.

Bacterial isolates were inoculated into the TSB medium supplemented with

tryptophan (50mg/ml) and inoculated at 28oC for 24hr. Cultures were centrifuged at

7000 rpm for 5 min at 4oC. The supernatant was collected and finally passed through

0.2 m Millipore filter and two drop of ortho-phosphoric acid was added to 2 ml of

supernatant to develop pink colour.

HCN production

The bacterial culture of Pseudomonas was streaked on TSM medium plates


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Pseudomonas Aeruginosa Strains and their Role 51

supplemented with 4.4g per litre glycine with simultaneously supplemented filter

paper soaked in a 0.5% picric acid in 1% Na2CO3 in the upper lid of petriplate. The

plates were sealed with paraffin and control plates did not receive any Pseudomonas

inoculum. Plates were incubated at 2810C for 1-2days. Change in colour of the filter

paper from yellow to brown, moderate brown to strong reddish brown indicated HCN

production.

Siderophore production

Pseudomonas strains were spread over tryptic soya agar medium and incubated at 28

10C for 24 h. Thereafter, a thin layer of CAS reagent in 0.7% agar was spread over

the colonies ofPseudomonas and plates were re-incubated at 28 10C for 24-48h.

Observe formation of yellow-orange halo around the colony shows siderophore

production.

P-solubilization test

Characterized Pseudomonas strains were transfer on Pikovskyas Agar medium and

inoculated at 281oC for 3-5days and clear zone around the colony showed P-

solubilization.

Pot experimentSeed bacterization:Mucuna seeds were surface-sterilized with 0.5% NaOCl solution

for 12 min, rinsed in sterilized distilled water and dried under a sterile air stream.

Cells ofPseudomonas strains were grown under continuous shaking condition (150rpm) on KingB broth at 28 1C for 24h. Each culture was separately centrifuged at

7000 rpm for 15 min at 4C. The culture supernatant was discarded and the pellets

were washed with sterile distilled water (SDW) and resuspended in SDW to obtain a

population density of 108

cfu ml-1

. The cell suspension was mixed with 1%

carboxymethylcellulose (CMC) solution. The slurry was coated separately on the

surface ofMucuna seeds and allowed to air-dry overnight in aseptic condition. The

seeds coated with 1% CMC slurry without bacterial strains served as control.

Pot size and soil: Sterile earthen pots (24 cm 12 cm 12 cm) were filled with

unsterilized sandy loam soil (0.25% total organic matter, 0.096% total organic C, 38%

water-holding capacity, pH 6.8).

Treatments: Total 06 treatment were prepared and these are Pseudomonas MR-2 +

seed, Pseudomonas MR-5 + seed, Pseudomonas MR-9 + seed, Pseudomonas MR-15

+ seed, Pseudomonas MR-18 + seed and and uninoculated seed (control). Four

bacterized seeds per pot were sown except control (non-bacterized seeds). After 15

days, thinning was done to raise only single healthy plant in each pot. The plants were

irrigated with sterilized water whenever required. Plant data such as plant dry weight,

plant height, number of nodule and nodule fresh weight per plant were recoded after

60 days of sowing. The data were analyzed statistically by using analysis of variance

(ANOVA).


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Results and DiscussionPseudomonas strains were isolated from rhizospheric soil ofMucuna plant. On kings

B agar medium, 20 fluorescent colonieswere separated and characterized on the basis

of various bio-chemicals test and results were compared with Bergeys Manual of

Determinative Bacteriology [12]. These characterized Pseudomonas aeruginosa

strains were selected for further study and renamed as MR-1 to MR-20. PGPR activity

were analysed and found some promising results.

Indole Acetic Acid (IAA) is plant growth hormones. IAA reacts with ortho-

phosphoric acid and produced pink colour. Such observation has been observed in

70% isolated Pseudomonas strains i.e IAA positive strains. In HCN test, all strains

except MR-1, MR-3, MR-7, MR-10, MR-14, MR-16, MR-17, MR-19 showed

changed in colour of filter paper from yellow to brown, which showed that strains

were HCN positive. Siderophore is iron chelating had been shown in 55% strainsPseudomonas strains. In siderophore positive strains showed yellow orange halo

around the colony. Clear zone around the colony of Pseudomonas strains on

Pikovskyas agar medium showed P-solubilization. 60% of isolated Pseudomonas

strains solubilized phosphorous. Only five strains MR-2, MR-5, MR-9, MR-15, MR-

18 produced siderophore, HCN, IAA and solubilised phosphorous (Table 1). Results

suggested that isolated Pseudomonas strains were plant growth promoting bacteria

(PGPB). Some other reports also supported our results. Recently Deshwal and Vig [8]

reported that PGPR strains produced Indole Acetic Acid (IAA), HCN, Siderophore, P-

solubilization. Similar to PGPR (particularly fluorescent pseudomonads) the

symbiotic rhizobacteria enhance plant growth due to IAA production [13] and also

solubilize insoluble inorganic phosphorus [1]. PGPR could directly enhance plantgrowth by phytohormones production and enhanced nutrient uptake [14].

Table 1: Production of IAA, HCN, siderophore and phosphate solubilization by

Pseudomonas aeruginosa strains.

Pseudomonas aeruginosa IAA HCN Siderophore P-solubilization

Pseudomonas MR-1 + - + +

Pseudomonas MR-2 + + + +

Pseudomonas MR-3 + - - +

Pseudomonas MR-4 - + - +


Pseudomonas MR-6 - + + -

Pseudomonas MR-7 + - + -

Pseudomonas MR-8 + + - +



Pseudomonas MR-11 - + - -

Pseudomonas MR-12 + + - -

Pseudomonas MR-13 - + - -


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Pseudomonas MR-14 - - + +



Pseudomonas MR-17 + - - +


Pseudomonas MR-19 + - - -

Pseudomonas MR-20 - + - +

Pseudomonas aeruginosa MR-9 enhanced maximum plant dry weight, plant

height, nodule per plant, nodule fresh weight per plant by 184, 124, 139, 180%

respectively as compared to control. Pseudomonas MR-2, MR-5, MR-15, MR-18

increased plant dry weight, plant height, nodule per plant, nodule fresh weight perplant by 172, 168, 112, 176 % respectively as compared to control. Similarly,

bacterized seeds promoted plant dry weight, plant height, nodule per plant, nodule

fresh weight per plant (Table 2). Nodulation data showed that Pseudomonas strains

improve nodule number and nodule weight. It seems that Pseudomonas strains

improved the Rhizobium population which was present in soil and PGPR activity

enhanced plant growth activity. Similar reports supported our results. PGPR enhanced

plant yield in a wide range of crop plants such as vegetables, rapeseed [15, 16], spring

and winter wheat by 8-16% [15], sugar beet to 7-10% [17] and in radish 60-144%

[18]. Our result suggested that Pseudomonas aeruginosa produce IAA, HCN,

Siderophore and P-Solubilization and as a result increased plant growth and

nodulation in medicinal plant Mucuna.

Table 2: Effect of bacterization with Pseudomonas aeruginosa strains on plant dry

weight, plant height, nodules per plant and fresh nodule weight of Medicinal plant

(Mucuna pruriens).

Treatment

number

Treatment Plant Nodule

Dry

weight**

(g)

Height**

(cm)

Number per

plant**

Fresh weight

per plant**

Treatment 1 Pseudomonas MR-

2 + Seed

4.3 376 39 1.46


5 + Seed

4.2 363 36 1.29


9 + Seed

4.6 392 46 1.96


15 + Seed

2.8 337 32 0.91


18 + Seed

4.4 374 37 1.47

Treatment 6 Control 2.5 316 33 0.92


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Values are mean of three replicates; ns- non-significant at 0.05 level of ANOVA *-

significant at 0.05 level of ANOVA, **- significant at 0.01levels of ANOVA.

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pseudomonas aeruginosa strains and their role in plant growth promotion in medicinal plant

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