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SHORT COMMUNICATION
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- vicinity [1]. The rhizosphere gives support to many active
eficial bacteria are one of the major groups of microbes that microbial populations capable of exerting beneficial, neu-
can be found in the rhizosphere, in association with roots tral or detrimental effects on plant growth [2]. Rhizobac-
which can enhance the growth of plant directly or indi- teria (root colonizing bacteria) that exert the beneficial
rectly. The present study aimed to isolate plant growth effects on the growth of the host plant via direct or indirect
promoting and disease suppressing bacterial isolates native mechanisms are termed as plant growth promoting rhi-
to rhizosphere soil and/or endophytic tissues of ginger zobacteria (PGPR) [3]. Indirect effects are related to pro-
plants from two different major growing location of each of duction of metabolites such as antibiotics, HCN, or
Solan and Sirmour districts of H.P. (India). Nutrient agar, siderophores. Direct effects are dependent on production of
Jensen’s medium, Pikovskaya’s medium and soil extract plant growth regulators, improvement in plant nutrients
medium are used for screening of microbial population uptake, promote induce systemic resistance (ISR) of the
associated with ginger and ginger roots, while for the plant [4], increasing plant health and soil fertility [5]. These
population assessment, Pikovskaya’s medium was used. mechanisms can be active simultaneously or independently
The highest microbial count on ginger was at Sirmour at different stages of plant growth [6]. Further bio-fertil-
(Narag), while the PSB ?ve colonies (87.72) and % P izers such as microbial inoculants promote plant growth,
solublizers to PVK count (79.00) associated with ginger productivity and increase the nutrient status of the host
root was highest at Solan (Kandaghat), Himachal Pradesh. plant have internationally been accepted as an alternative
source of chemical fertilizers [7]. Solan and Sirmour are
Keywords Zingiber officinale Isolation Bacillus spp. the major ginger growing districts of Himachal Pradesh in
Plant growth promoting rhizobacteria 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
Microbial communities are copiously present in rhizo- of organic production of crops, the application of PGPR is
sphere or areas under the influence of the root and its close 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
K. Kaundal R. Kaushal S. Gupta to isolate, enumerate and characterize various PGPR’s
Department of Basic Science, College of Forestry, Dr. Y.S. from the rhizosphere of ginger.
Parmar University of Horticulture and Forestry, Nauni, Solan
Rhizosphere soil samples were collected from different
173 230, Himachal Pradesh, India
e-mail: kirtikndl@gmail.com locations of ginger growing fields in Solan (Kandaghat and
Nauni) and Sirmour (Narag and Rajgarh) districts of
K. Sharma (&) Himachal Pradesh. From each district, two sites and two
Department of Food Science and Technology, College of
sub-sites were selected for sampling purpose. The samples
Horticulture, Dr. Y.S. Parmar University of Horticulture and
Forestry, Nauni, Solan 173 230, Himachal Pradesh, India were placed in plastic bags and stored for further isolation
e-mail: kajalmcro20@gmail.com and analysis work. Modified replica plating technique was
123
54 K. Kaundal et al.
used to isolate PGPR isolates from rhizosphere and roots of isolation of PGPR’s isolates. Microorganism population in
ginger. One gram of rhizosphere soil was placed in 9 ml of rhizosphere and roots of ginger at different locations/sites
sterilized distilled water under aseptic conditions. The were presented in Table 1. The total microbial counts, in
suspension of soil was spread on pre-poured nutrient agar general, were more in rhizosphere
5
(NA) medium. After incubation of 24–48 h, the isolated (279.69–252.77 9 10 cfu/g soil) as compared to endo-
colonies that developed on enriched medium (master phytic (111.70–103.33 9 101 cfu/g root) counts. Among
replica) were replica plated on to the selective media: various sites, Narag (Sirmour) had the highest
Nitrogen free medium [11] for nitrogen fixing activity, (222.40 9 105 cfu/g soil) bacterial counts on NA medium
Pikovskaya medium [12] for phosphate solubilising ability. and also the highest microbial population on soil extract
All colonies were transferred to same position as the master medium (279.69 9 105 cfu/g soil). The population on the
plate with the help of wooden block covered with sterilized Jensen’s medium which is specific for asymbiotic N-fixers
velvetin cloth. At the end of the incubation period, the was highest (75.34 9 105 cfu/g soil) at Narag (Sirmour)
location of the colonies appeared on the replica plates were while the population on the Pikovskaya’s medium specific
compared to the master plate. The microbial count was for P-solubilisation was highest (82.35 9 105 cfu/g soil) at
expressed as colony forming units (cfu) per gram of soil. Nauni (Solan). It has been reported that higher concentra-
For isolation of endophytic rhizobacteria, the root sample tions of phosphate-solubilizing bacteria are commonly
was surface sterilized by 0.2 % mercuric chloride (HgCl2) found in the rhizosphere soil as compared to non-rhizo-
for two minutes followed by repeated washing in sterilized spheric soil [17]. The endophytic bacterial population was
distilled water. The surface sterility of rhizome was cross highest on NA medium (116.25 9 101 cfu/g roots) from
checked by incubating the surface sterilized rhizome in Nauni (Solan) region. The maximum (52.32 9 101 cfu/g
sterilized NA medium for 24 h at 35 ± 2 °C. One gram of roots) endophytic bacterial population capable of N-fixa-
surface sterilized rhizome sample was crushed in 9 ml of tion as shown by their growth on Jensen’s medium was
sterilized distilled water and was crushed to produce slurry recorded for Rajgarh (Sirmour). P-solubilization was
using pestle and mortar under aseptic conditions. The recorded highest endophytic bacterial counts
serially diluted suspension of soil was spread on pre-poured (65.98 9 101 cfu/g roots) at Narag (Sirmour). These
NA medium. Rest of process remains same as that for results are in proximity with the studies of [18], who
isolation of rhizobacteria and the results were expressed in reported that under natural conditions, the rhizosphere and
cfu/gram of root. rhizoplane of the plants harbor a large and varied popula-
For preparation of inoculum, a bacterial cell suspension tion of the microorganisms. The variation in microbial
(OD 1 at 540 nm) of 48 h old culture grown on nutrient population both in rhizosphere and roots may be attributed
broth at the rate of 10 % was used as inoculum in all to climatic conditions of the location, age of plant, variety/
experiments. Turbidity/growth was monitored by measur- cultivar type, time of sampling and physico–chemical
ing the change in absorbance of cells in the broth at 540 nm properties of soil. The results are further in line with those
using un-inoculated broth as blank. of [19] who has also reported variation in microbial pop-
The screening of the bacterial isolates for various plant ulation with respect to location/plant parts. Different
growth promoting activities like P-solubilization [12] environment parameters, content of soil organic carbon,
siderophore [13], HCN [14] growth on N-free medium, total nitrogen and altitude could affect the diversity of soil
auxin production and antagonism against Pythium spp. and flora [20]. Endophytic bacteria reside within the plant tis-
Fusarium spp. were performed by adopting the standard sues and thus have a natural and intimate association with
methods. The brief description of these methods is as; (1) plants. The internal tissues of plants provide relatively
Phosphate solubilizing activity was measured on PVK uniform and protected environment when compared with
medium [12] while nitrogen fixing activity was observed in rhizosphere and rhizoplane [21].
a straight line on Jensen’s medium. Siderophore production The data on wealth and diversity of the population of
was detected by CAS plate assay method [14]. Quantitative P-solubilizers in rhizosphere and roots for different loca-
estimation of indole-3-acetic acid was measured by the tions/sites are also presented in Table 1. The percentage of
method described by Gorden and Palleg [15]. Antagonistic phosphate solubilizing microorganisms to total PVK count
activity of bacterial isolates against test fungus (Pythium ranged from 63.88 (Nauni, Solan) to 78.36 % (Rajgarh,
spp. and Fusarium spp.) (Fig. 1) was calculated as Sirmour). Table further clearly showed that per cent
described by Vincent [16]. phosphate solubilizers to the total population on PVK
The rhizosphere serves as a very dynamic habitat for medium at different location/sites varied from 62.42 at
microorganisms. The ginger rhizospheric and roots/rhi- Narag (Sirmour) to 79.00 % at Kandaghat (Solan). Popu-
zome samples were collected from its natural habitat for lation of phosphate solubilizing microorganisms, in
123
Isolation of Plant Growth from Z. officinale 55
123
56 K. Kaundal et al.
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
[24].
Medium
(PVK)
56.34
59.27
62.32
65.98
References
Microbial count (9101 cfu/g roots)
Pikovskaya’s
population associated with ginger
56.34
59.27
62.32
65.98
BeruEcksichtigung der GruEnduEngung und Brache. Arbeit-
Enumeration of rhizosphere bacterial population Population of P-solubilizers associated Enumeration of microbial
Jensen’s
medium
agar
28(4):1327–1350
colonies to PVK
pp 886–889
with ginger
Narag
biol 43(10):895–914
123
Isolation of Plant Growth from Z. officinale 57
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