PHARMACEUTICAL AND BIOLOGICAL EVALUATIONS

June 2016; vol. 3 (Issue 3): 371-376.

www.onlinepbe.com ISSN 2394-0859

Research Article

Studies on biosynthesis of auxin in rhizobium and their impact

on growth of Vigna mungo L.
Parthiban P.1, Shijila Rani A.S.1, Mahesh V.2, Ambikapathy V.3*
1
Department of Microbiology, Marudupandiyar College, Vallam, Thanjavur, Tamil Nadu, India
2
MR Arts and Science College, Mannargudi, Tamilnadu, India
3
Department of Botany and Microbiology, AVVM Sri Pushpam College, Poondi, Tamil Nadu, India

*For correspondence ABSTRACT

Ambikapathy V.,
Department of Botany and
Microbiology, AVVM Sri
Pushpam College, Poondi,
Tamil Nadu, India.
Email: drva1967@gmail.com
Received: 21 April 2016
Revised: 02 May 2016
Accepted: 11 May 2016
Objective: The present investigation, the use of plant growth promoting
rhizobacteria was used to growth and improves the yield in crop of Vigna
mungo.
Methods: Rhizobium strains were isolate and identified by Yeast Extract
Mannitol Agar medium with congored (CYEMA) and some biochemical
test were performed. Separation and quantification of growth hormone by
paper chromatography and spectrophotometric methods followed and its
impact of Vigna mungo in green house experiments.
Results: The Rhizobium strains were isolated from legumes plant Mimosa
pudica L, Arachis hpogea L and Vigna mungo L. Indole-3-acetic acid
(IAA) produced from the L-trptophan medium and identified by using
paper chromatography techniques. The pot culture experiments were
conducted with various treatment of Rhizobium sp, strains are high
growth and yield.
Conclusions: Rhizobium is beneficial organisms and to interaction with
growth promotion to all leguminous plants as well as nitrogen fixation,
its biological process of the ecosystem. It is concluded to the Rhizobium
species is eco-friendly to farmer and these environments.
Keywords: Root nodules, Rhizobium, IAA, Vigna mungo L. seeds

Introduction in vicinity of the root. It has been recongnized

Growth hormones are substance synthesized in
particular cells, in extremely small quantitive
influence developmental process. Plant growth
hormones was vital role in controlling plant
growth such as, Auxins, gibberllins, cytokines,
ethylene, and abiscissic acid, they are naturally
synthesized by some microbes. Bacteria are
abundantly present in the rhizosphere and close
that several generea of these rhizobacteria have
the ability to promote plant growth and these
have been termed plant growth promoting
rhizobacteria or PGPR.1
Rhizobia are well known for their capacity to
establish a symbiosis with legumes. The
interaction between Rhizobium bacteria and
leguminous plants results in nodule formation
and atmospheric nitrogen is converted to

Pharmaceutical and Biological Evaluations 371

Parthiban P. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (3): 371-376.
ammonia and then metabolized by the plant2.
They also produce plant hormones like auxins,
gibberellins cytokinines, ethylene, and abscisic
acid in addition to symbiotic nitrogen fixation.
Among the phytohormones, auxin was the first
plant hormone discovered.2 Several workers
reported that Rhizobium sp. produced high
amount of auxin in culture with or without L-
tryptophan in the YEMA medium.
The phyto hormones auxin is synthesized in the
shoot tips of growing plants. The growth
hormone auxin was elongation growth, cell
division and differentiation and apical
dominance. Zinc is essential element for auxin
biosynthesis. The highest concentrations of
auxin were found in growing tips, buds, leaves
and roots of the plants.
Materials and Methods
Sample collection: Root nodules were collected
from young and healthy seedling plants of
Mimosa pudica L, Arachis hpogea L and Vigna
mungo L plant from Oruthanadu, Thanjavur
(dist), Tamil nadu, India.
Isolation of Rhizobium sp from root nodules:
To select, healthy and reddish-pink color root
nodules were tenderly washed with distilled
water and surface sterilized by keeping in 0.1
percent HgCl2 for 4-5 min and wash with sterile
distilled water followed by 95 percent ethyl
alcohol, repeat washing with sterile distilled
water.3 The nodules were crushed in a small
drop of sterile distilled water with the help of
sterile glass rod and obtain milky suspension.
Then, serial dilutions were made and aliquots
dilutions were spread on yeast extract mannitol
agar (YEMA) medium and plates were
incubated at 281C.4
Identification and biochemical test of
Rhizobium sp: The isolated colonies were
confirmed by microscopic observation and
biochemical test6,7 and cultural test were
performed. Isolates were identified as per
Bergeys manual of Systematic bacteriology.
Biosynthesis of IAA: Then, the colonies were
grown individually in trypticase soya broth
(TSB) for 30 min, and one ml of cell free culture
Pharmaceutical and Biological Evaluations
filtrate to each strain was added to two ml of
Salkowaski reagent and incubated at 282C for
48 hours in shaking incubator at 1000 rpm. The
content was filtered through Whatmann No: 1
filter paper before measuring IAA in terms the
broth, these contents was determined by
colorimetrically at 530 nm.8
Identification of IAA: Biosynthesized auxins
were partially characterized by paper
chromatography method.9 In which three, grams
of culture filtrate were extracted with 50 ml of
peroxide-free ether for 2 hours at 5oC. The
appearance of the strips under ultraviolet light
and spraying with modified Salkowski reagent is
indicated.
Pot culture method: The pot trails were
accomplished by Department of Microbiology
Marudupandiyar College, Vallam, Thanjavur.
Each one had conducted triplicates. The
treatment follows as:
T1 Control (without Rhizobium); T2 Rhizobium
(R1 seed inoculation); T3 Rhizobium (R2 seed
inoculation); T4 Rhizobium (R3 seed
inoculation); T5 Rhizobium (R1 seed inoculation
+foliar spray); T6 Rhizobium (R2 seed
inoculation + foliar spray); T7 Rhizobium (R3
seed inoculation + foliar spray).
After germination, plants were treated with 48
hrs old culture, which were foliar sprayed to the
Vigna mungo plants by every week, starting
from 10th day of germination. The plants were
harvested every 15th day of intervals analyze
morphometric, biometric and number of root
nodules were analyzed.
Estimation of carbohydrates10: Fresh leaves 100
mg was taken in a test tube and hydrolysed with
2 ml of concentrated H2SO4 for 30 minutes at
100C. To 0.5 ml of 5 percent phenol and 5 ml
of conc. H2SO4 were added and mixed
thoroughly. The colour developed was measured
at 490 nm in Spectronic 20. Glucose was used as
a standard.
Estimation of total proteins11: 100 mg of fresh
leaves was ground in mortar and pestle with 1.0
ml of phosphate buffer and centrifuged at
10,000x g for 10 minutes. To 0.5 ml and 5 ml of
372
Parthiban P. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (3): 371-376.

alkaline copper reagent were added and allowed Table 2: Quantitative assessment of growth
to stand for 10 minutes, finally 0.5 ml Folins hormone production.
reagent was added. The absorbance was
measured after 30 minutes at 750 nm in S.No Treatment IAA(mg/ml)
spectronic 20. The standard graph was prepared 1 R1 3.00
by using Bovin Serum Albumin. 2 R2 3.20
3 R3 2.02
Results and Discussion 16
Total
14 carbohydrate
In the present investigation, the isolates of 12 (mg) 15th

OD at490 nm
rhizobia from root nodules of Arachis hypogea 10 day

and Vigna mungo plants were characterized by 8 Total

opaque and milky white in appearance in YEMA 6 carbohydrate
(mg) 30th
medium. The isolated rhizobial strains were 4
day
identified in microscopic and biochemical test to 2
0
perceive as positive results (Table 1). All strains T1 T2 T3 T4 T5 T6 T7
were gram negative and did not absorb red color
when cultured in YEMA containing congo red. Figure 1: Effect of rhizobia on carbohydrate
The result were compared with Bergeys manual content of Vigna mungo.
of Determinative Bacteriology and conformed as
35
Rhizobium. These Rhizobial isolates produced Total
auxins in-vitro conditions in the ranges from 30
protein(m
3.00 to 3.8 mg/L, IAA equivalents without L- 25 g)
15thday
OD at 750 nm

tryptophan. The pot trial was conducted to study 20

Total
the response of Vigna mungo to inoculation with 15
protein(m
rhizobium and foliar application. The result 10 g) 30th
day
revealed that the black gram responded well to 5
the inoculation of rhizobium. The plants 0
T1 T2 T3 T4 T5 T6 T7
inoculated with native rhizobium possessed
significantly greater shoot, root height, dry
weight, nodule numbers, total carbohydrate and Figure 2: Effect of rhizobia on protein
protein and nodules formation was monitor and content of Vigna mungo.
tabulated (Table 3, 4, Figure 1 and 2).

Table 1: Microscopic and biochemical
characterization of Rhizobial isolates.
S. No Characteristics
1 Gram staining
2 Motility
3 Indole test
4 Methyl red test
5 Voges-Proskauer test
6 Citrate utilization
7 Starch hydrolysis
8 Urea hydrolysis
9 H2S production
10 Catalase test
11 Lactose
12 Maltose
Shahzad et al.12 isolated Rhizobium from root
nodules of Alfalfa (Medico sativa) plant and
characterized on the basis of various
Results biochemical tests. Previously, Ashfaq et al.13 the
- rhizobial strains were isolated from the nodules
+ of Mung bean and Pisum sativum.14 Similarly,
+ Singh et al.15 also characterized Rhizobium
- strains on the basis of biochemical tests.
- Rhizobium is symbiotic bacteria which form
+ nodule in leguminous plant. Auxin production is
- frequently pointed out.13,16-20 Different legume
+ nodulating rhizobial strains preferred different
- vitamins sources for IAA production
+ reported.21,22 The current observations of the pot
- experiment of pea was close to Fischer et al.23
+ who find out the ability of Rhizobium on wheat
by development of shoot/ root fresh and dry

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Parthiban P. et al. Pharmaceutical and Biological Evaluations 2016; vol. 3 (3): 371-376.

Table 3: Effect of Rhizobium on plant height and biomass of Vigna mungo from 15th days of treated
with inoculums.

S. No Treatment Plant height (cm) Plant dry weight No. of

mg/plants nodules
shoot root
1 T1 16.5 4.5 85.0 -
2 T2 20.5 5.1 139.0 -
3 T3 20.5 6.7 137.0 -
4 T4 20.5 5.5 124.7 -
5 T5 20.3 6.5 123.7 -
6 T6 23.3 5.5 83.0 -
7 T7 20.3 5.7 125.4 -
T1 Control (without Rhizobium); T2. Rhizobium (R1 seed inoculation); T3 Rhizobium (R2 seed inoculation); T4 Rhizobium (R3
seed inoculation); T5 Rhizobium (R1 seed inoculation +foliar spray); T6 Rhizobium (R2 seed inoculation + foliar spray); T7
Rhizobium (R3 seed inoculation + foliar spray)

Table 4: Effect of Rhizobia on plant height and biomass of Vigna mungo after 30 day treatment.

S. No Treatment Plant height (cm) Plant dry weight No. of

shoot root mg/plants nodules
1 T1 18 6.4 89.0 -
2 T2 22 8 145.0 3
3 T3 22.5 7.2 143.0 3
4 T4 22 7 130.7 4.2
5 T5 23 10 131.0 -
6 T6 25 8 92.0 -
7 T7 19 17 130.4 -
T1 Control (without Rhizobium); T2. Rhizobium (R1 seed inoculation); T3 Rhizobium (R2 seed inoculation); T4 Rhizobium (R3
seed inoculation); T5 Rhizobium (R1 seed inoculation +foliar spray); T6 Rhizobium (R2 seed inoculation + foliar spray); T7
Rhizobium (R3 seed inoculation + foliar spray)

weights. All the Rhizobium strains improved the
root and shoot dry biomass by 100% and 70%
respectively. Some workers reported in who
obtained 70% increase in pea root/shoot dry
biomass by PGPRs inoculation as contrast to
control (uninoculated).24
Conclusions
From this study, finish off that the rhizobia was
the most common plant growth promoting
bacteria to produce plant growth promoting
hormones of auxin. Arachis hypogia isolate can
be promoting early blackgram growth, besides
increasing plant height, dry weight, total
carbohydrate and protein. However, further
studies are needed for an integrative
understanding of hormones and metabolism in
Rhizobium.
Acknowledgements
The authors wish to thankful of Mardupandiyar
College, Vallam, Thanjavur, for extending the
facilities to carry out the laboratory studies.
Funding: No funding sources
Conflict of interest: None declared

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