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Isolation of Plant Growth Promoting Rhizobacteria from Ginger (Zingiber

officinale Rosc.) Rhizome for Future Studies

Article  in  National Academy Science Letters · January 2016

DOI: 10.1007/s40009-015-0367-3

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Natl. Acad. Sci. Lett. (January–February 2016) 39(1):53–57
DOI 10.1007/s40009-015-0367-3
SHORT COMMUNICATION
Isolation of Plant Growth Promoting Rhizobacteria from Ginger
(Zingiber officinale Rosc.) Rhizome for Future Studies
Kirti Kaundal • Rajesh Kaushal • Kajal Sharma
Shweta Gupta
Received: 22 March 2014 / Revised: 2 August 2014 / Accepted: 6 January 2015 / Published online: 13 January 2016
Ó The National Academy of Sciences, India 2016
Abstract Plant growth promoting rhizobacteria are ben-
eficial bacteria are one of the major groups of microbes that
can be found in the rhizosphere, in association with roots
which can enhance the growth of plant directly or indi-
rectly. The present study aimed to isolate plant growth
promoting and disease suppressing bacterial isolates native
to rhizosphere soil and/or endophytic tissues of ginger
plants from two different major growing location of each of
Solan and Sirmour districts of H.P. (India). Nutrient agar,
Jensen’s medium, Pikovskaya’s medium and soil extract
medium are used for screening of microbial population
associated with ginger and ginger roots, while for the
population assessment, Pikovskaya’s medium was used.
The highest microbial count on ginger was at Sirmour
(Narag), while the PSB ?ve colonies (87.72) and % P
solublizers to PVK count (79.00) associated with ginger
root was highest at Solan (Kandaghat), Himachal Pradesh.
Keywords Zingiber officinale
Isolation
Bacillus spp.

Plant growth promoting rhizobacteria
Microbial communities are copiously present in rhizo-
sphere or areas under the influence of the root and its close
K. Kaundal
R. Kaushal
S. Gupta
Department of Basic Science, College of Forestry, Dr. Y.S.
Parmar University of Horticulture and Forestry, Nauni, Solan
173 230, Himachal Pradesh, India
e-mail: kirtikndl@gmail.com
K. Sharma (&)
Department of Food Science and Technology, College of
Horticulture, Dr. Y.S. Parmar University of Horticulture and
Forestry, Nauni, Solan 173 230, Himachal Pradesh, India
e-mail: kajalmcro20@gmail.com
•
vicinity [1]. The rhizosphere gives support to many active
microbial populations capable of exerting beneficial, neu-
tral or detrimental effects on plant growth [2]. Rhizobac-
teria (root colonizing bacteria) that exert the beneficial
effects on the growth of the host plant via direct or indirect
mechanisms are termed as plant growth promoting rhi-
zobacteria (PGPR) [3]. Indirect effects are related to pro-
duction of metabolites such as antibiotics, HCN, or
siderophores. Direct effects are dependent on production of
plant growth regulators, improvement in plant nutrients
uptake, promote induce systemic resistance (ISR) of the
plant [4], increasing plant health and soil fertility [5]. These
mechanisms can be active simultaneously or independently
at different stages of plant growth [6]. Further bio-fertil-
izers such as microbial inoculants promote plant growth,
productivity and increase the nutrient status of the host
plant have internationally been accepted as an alternative
source of chemical fertilizers [7]. Solan and Sirmour are
the major ginger growing districts of Himachal Pradesh in
India, as a rain fed crop [8]. Soils of these districts are, in
general, deficient in nitrogen and having problems of
availability of phosphorus nutrition. In the present scenario
of organic production of crops, the application of PGPR is
the effective, ecofriendly, low volume, supplementing
source of nutrients, not only for increasing crop yields but
also to sustain soil health [9, 10]. The aim of the study was
to isolate, enumerate and characterize various PGPR’s
from the rhizosphere of ginger.
Rhizosphere soil samples were collected from different
locations of ginger growing fields in Solan (Kandaghat and
Nauni) and Sirmour (Narag and Rajgarh) districts of
Himachal Pradesh. From each district, two sites and two
sub-sites were selected for sampling purpose. The samples
were placed in plastic bags and stored for further isolation
and analysis work. Modified replica plating technique was
123
54
used to isolate PGPR isolates from rhizosphere and roots of
ginger. One gram of rhizosphere soil was placed in 9 ml of
sterilized distilled water under aseptic conditions. The
suspension of soil was spread on pre-poured nutrient agar
(NA) medium. After incubation of 24–48 h, the isolated
colonies that developed on enriched medium (master
replica) were replica plated on to the selective media:
Nitrogen free medium [11] for nitrogen fixing activity,
Pikovskaya medium [12] for phosphate solubilising ability.
All colonies were transferred to same position as the master
plate with the help of wooden block covered with sterilized
velvetin cloth. At the end of the incubation period, the
location of the colonies appeared on the replica plates were
compared to the master plate. The microbial count was
expressed as colony forming units (cfu) per gram of soil.
For isolation of endophytic rhizobacteria, the root sample
was surface sterilized by 0.2 % mercuric chloride (HgCl2)
for two minutes followed by repeated washing in sterilized
distilled water. The surface sterility of rhizome was cross
checked by incubating the surface sterilized rhizome in
sterilized NA medium for 24 h at 35 ± 2 °C. One gram of
surface sterilized rhizome sample was crushed in 9 ml of
sterilized distilled water and was crushed to produce slurry
using pestle and mortar under aseptic conditions. The
serially diluted suspension of soil was spread on pre-poured
NA medium. Rest of process remains same as that for
isolation of rhizobacteria and the results were expressed in
cfu/gram of root.
For preparation of inoculum, a bacterial cell suspension
(OD 1 at 540 nm) of 48 h old culture grown on nutrient
broth at the rate of 10 % was used as inoculum in all
experiments. Turbidity/growth was monitored by measur-
ing the change in absorbance of cells in the broth at 540 nm
using un-inoculated broth as blank.
The screening of the bacterial isolates for various plant
growth promoting activities like P-solubilization [12]
siderophore [13], HCN [14] growth on N-free medium,
auxin production and antagonism against Pythium spp. and
Fusarium spp. were performed by adopting the standard
methods. The brief description of these methods is as; (1)
Phosphate solubilizing activity was measured on PVK
medium [12] while nitrogen fixing activity was observed in
a straight line on Jensen’s medium. Siderophore production
was detected by CAS plate assay method [14]. Quantitative
estimation of indole-3-acetic acid was measured by the
method described by Gorden and Palleg [15]. Antagonistic
activity of bacterial isolates against test fungus (Pythium
spp. and Fusarium spp.) (Fig. 1) was calculated as
described by Vincent [16].
The rhizosphere serves as a very dynamic habitat for
microorganisms. The ginger rhizospheric and roots/rhi-
zome samples were collected from its natural habitat for
123
K. Kaundal et al.
isolation of PGPR’s isolates. Microorganism population in
rhizosphere and roots of ginger at different locations/sites
were presented in Table 1. The total microbial counts, in
general, were more in rhizosphere
5
(279.69–252.77 9 10 cfu/g soil) as compared to endo-
phytic (111.70–103.33 9 101 cfu/g root) counts. Among
various sites, Narag (Sirmour) had the highest
(222.40 9 105 cfu/g soil) bacterial counts on NA medium
and also the highest microbial population on soil extract
medium (279.69 9 105 cfu/g soil). The population on the
Jensen’s medium which is specific for asymbiotic N-fixers
was highest (75.34 9 105 cfu/g soil) at Narag (Sirmour)
while the population on the Pikovskaya’s medium specific
for P-solubilisation was highest (82.35 9 105 cfu/g soil) at
Nauni (Solan). It has been reported that higher concentra-
tions of phosphate-solubilizing bacteria are commonly
found in the rhizosphere soil as compared to non-rhizo-
spheric soil [17]. The endophytic bacterial population was
highest on NA medium (116.25 9 101 cfu/g roots) from
Nauni (Solan) region. The maximum (52.32 9 101 cfu/g
roots) endophytic bacterial population capable of N-fixa-
tion as shown by their growth on Jensen’s medium was
recorded for Rajgarh (Sirmour). P-solubilization was
recorded highest endophytic bacterial counts
(65.98 9 101 cfu/g roots) at Narag (Sirmour). These
results are in proximity with the studies of [18], who
reported that under natural conditions, the rhizosphere and
rhizoplane of the plants harbor a large and varied popula-
tion of the microorganisms. The variation in microbial
population both in rhizosphere and roots may be attributed
to climatic conditions of the location, age of plant, variety/
cultivar type, time of sampling and physico–chemical
properties of soil. The results are further in line with those
of [19] who has also reported variation in microbial pop-
ulation with respect to location/plant parts. Different
environment parameters, content of soil organic carbon,
total nitrogen and altitude could affect the diversity of soil
flora [20]. Endophytic bacteria reside within the plant tis-
sues and thus have a natural and intimate association with
plants. The internal tissues of plants provide relatively
uniform and protected environment when compared with
rhizosphere and rhizoplane [21].
The data on wealth and diversity of the population of
P-solubilizers in rhizosphere and roots for different loca-
tions/sites are also presented in Table 1. The percentage of
phosphate solubilizing microorganisms to total PVK count
ranged from 63.88 (Nauni, Solan) to 78.36 % (Rajgarh,
Sirmour). Table further clearly showed that per cent
phosphate solubilizers to the total population on PVK
medium at different location/sites varied from 62.42 at
Narag (Sirmour) to 79.00 % at Kandaghat (Solan). Popu-
lation of phosphate solubilizing microorganisms, in
Isolation of Plant Growth from Z. officinale 55

Fig. 1 Antagonistic activity of bacterial isolates against Pythium spp.

123
56 K. Kaundal et al.

Population of P-solubilizers associated

general, varied from 20 to 24 % of the total population and

solubilizer
in some soils it may be up to 85 % of the total population

colonies to PVK

77.11
79.00
64.29
62.42
count
[22]. In other studies conducted by [23] reported about

%P
16 % of the total bacterial population in rhizosphere was
P-solubilizers. The solubilization of phosphorous in the

61.98
87.72
73.97
61.98
PSB
?ve
rhizosphere is the most common mode of action implicated
with ginger roots

in PGPR that increase nutrient availability to host plants

Pikovskaya’s

[24].
Medium
(PVK)

56.34
59.27
62.32
65.98
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