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Australian Journal of Basic and Applied Sciences, 6(9): 183-187, 2012
ISSN 1991-8178
Effect of coinoculation of Mesorhizobium cicer with PGPR on Cicer arietinum
Amit Ladwal, Divya Bhatia and Deepak Kumar Malik
Department of Biotechnology Engineering, University Institute of Engineering & Technology,
Kurukshetra University, 136119, Haryana, India
Abstract: In this study chlorpyrifos degrading DKC2 and malathion degrading DKM3 DKM5, DKM8,
DKM9 bacterial strain were taken. The culture DKC2 showed siderophore production and strong plant
growth promoting activity when coinoculated with Mesorhizobium cicer Ca181. The strain DKC2
caused maximum gain in plants dry weight ratio 3.98 and 3.75 times when concoculated with
Mesorhizobium at 90 and 120 days respectively as compare to control. Thus the bacterial strain DKC2
can be used for plant growth promotion and as a biofertilizers to provide Fe nutrition to plants. Since
bacterium showing wide application in agro biotech with its pesticide degradation and plant growth
promoting activity more characterization of this bacterial strain is needed for its commercialization.
Key words: Coinoculation, Mesorhizobium cicer, PGPR, Cicer arietinum, Plant growth
INTRODUCTION
Plant growth-promoting rhizobacteria (PGPR) are free-living soil-borne bacteria that colonize the
rhizosphere, and when applied to seed or crops enhance the growth of plants (Kloepper 1980). PGPR enhance
plant growth either by direct or indirect mechanisms (Glick 1995). Bertrand et al. (2001) identified bacteria
belonging to the genera Pseudomonas, Agrobacterium and Phyllobacterium as the most efficient PGPR
associated with canola. The different microbial populations interact with each other and with the plant- microbe
interactions, which are useful, neutral or harmful, have been found to influence plant growth accordingly
(Astrom et al. 1993). The detrimental environmental impact of chemical fertilizers and their rising costs, the use
of PGPB as natural fertilizers is beneficial for the development of sustainable agriculture. PGPR promote plant
growth by various mechanisms that includes: (i) The ability to produce indole acetic acid (Suresh et al. 2010),
gibberelic acid (Mahmoud et al. 1984), cytokines (Tien et al. 1979) and ethylene (Glick 1995); (ii) symbiotic
nitrogen fixation (Hayat et al. 2008a); (iii) asymbiotic nitrogen fixation (Barka et al. 2006); (iv) antagonisms
against phytopathogenic microorganisms by the production of siderophores (Suresh et al. 2010), β-1,3glucanase (Stephane et al. 2005), chitinase (Frankowski et al. 2001), and antibiotics (Nakayama et al. 1999).
Bioremediation is the application of biological processes for the cleanup of hazardous chemicals present in the
environment (Gianfreda and Rao 2004). It has advantages over physicochemical methods due to several merits:
cost-effective, convenient, complete degradation of organic pollutants and no collateral destruction of the site
material or its indigenous flora and fauna (Timmis and Pieper 1999). The widespread use of PGPR for the
environmental remediation is a promising field (Lucy et al. 2004). It requires improving the effectiveness of too
little amounts of external inputs by employing the best combinations of beneficial bacteria in sustainable
agriculture production systems. Therefore, the present study was undertaken with the following objective:
detection of siderophore production and coinoculation of PGPR strains with Mesorhizobium sp. Cicer strain
Ca181in chickpea under pot house conditions
MATERIALS AND METHODS
Chemicals and Microorganisms:
The chemicals, media and reagents used for the present studies were from Hi Media Laboratories, SRL,
Glaxo and E. Merck etc. The chemicals were of AR grade. Standard culture of Mesorhizobium sp. strain Cicer
Ca181 used in the present studies was taken from the Department of Microbiology, CCS Haryana Agricultural
University and maintained on Luria Bertani medium. Seeds of chickpea (Cicer arietinum) var. HC3/8618 were
obtained from Pulses section, Department of Seed Technology, CCSHAU, Hisar. Different culture of PGPR
(DKC2, DKM3, DKM5, DKM8 and DKM9) used in the present studies was taken from the Department of
Biotechnology, UIET, Kurukshetra University and maintained on nutrient agar medium. The bacterial culture
DKC2 was responsible for chlorpyrifos degradation with some plant growth promoting activity (IAA). The
bacterial cultures (DKM3, DKM5 and DKM8) were responsible to utilized of malathion as a sole carbon source
with some plant growth promoting activity.
Corresponding Author: Deepak Kumar Malik, Department of Biotechnology Engineering, University Institute of
Engineering & Technology, Kurukshetra University, 136119, Haryana, India
E-mail: [email protected]
183 Aust. J. Basic & Appl. Sci., 6(9): 183-187, 2012
Detection and Nature of Siderophore:
The detection of siderophore in PGPR strains was done by using the method of Schwyn and Neilands
(1987). To check the nature of siderophore, the strain DKC2 was grown in culture media (Peptone 20 g, K2HPO4
1.5 g, MgSO47H2O 1.5 g, glycerol 10 ml in 1000 ml distilled water). After 48 h culture medium was centrifuged
to obtained cell free supernatant, then takes 1 ml of supernatant and adds few drops of FeCl3, OD was taken by
using spectrophotometer. Cell free supernatant without FeCl3 was used as a control.
Effect of PGPR Strains on Seedling Growth of Chickpea:
Healthy seeds of chickpea var. HC3/8618 were surface sterilized with acidic alcohol (H2SO4: ethanol, 7: 3
v/v) for 3 minutes followed by six thorough washings with sterilized water. The surface sterilized seeds were
inoculated with broth cultures of PGPR strains (DKC2, DKM3, DKM5, DKM8, and DKM9) for 30 min. The
inoculated seeds were germinated on plain water agar (0.8 %) at 28±1 °C. Uninoculated seeds treated with LB
broth were sown on agar plates as controls. Effects of inoculation on root and shoot length were measured at 8
and 15 days of growth.
Coinoculation of PGPR Strains with Mesorhizobium sp. cicer Strain Ca181 in Chickpea Under Pot House
Condition:
Chickpea var. HC3/8618 was used for symbiotic performance; healthy seeds were surface sterilized with
acidic alcohol. Surface sterilized seed were inoculated with broth culture of Mesorhizobium sp. cicer stain
Ca181 alone and as coinoculated by mixing with PGPR strains in a ratio of 1:1 (v/v). The 2 ml mixed inoculum
was used for 15 seeds and culture was allowed to be adsorbed on the seeds for half an hour. In case of strain
Ca181, 1 ml water was added to have relatively same level of inoculums. Control treatment without inoculation
was also kept. Seeds sown in sterilized pot, after germination only three healthy seedlings were kept. The pots
were put in pothouse under day light conditions. The plants were uprooted after 90 and 120 days to determine
the plant dry weight. For preparing pot: The pot and soil was autoclaved separately at 15 Ibs for 3 hr. After
autoclave the soil was filled in pot up to 2/3 portion. The treatments were as following: (A) Seed (without
inoculation), (B) Seed + Mesorhizobium, (C) Seed + DKC2, (D) Seed + DKM3, (E) Seed + DKM5, (F) Seed +
DKM8, (G) Seed + DKM9, (H) Seed + Mesorizobium + DKC2, (I) Seed + Mesorizobium + DKM3, (J) Seed +
Mesorizobium+DKM5, (K) Seed + Mesorizobium +DKM8, (L) Seed + Mesorizobium+ DKM9
Results:
Detection and Nature of Siderophore Production by PGPR:
Out of five, only two strains DKC2 and DKM5 were found to produce siderophore on CAS medium plates
as shown in Fig. 1. The CAS assay is a functional assay based on the high affinity of siderophores for iron and is
independent of their structural classification. When a siderophore removes the iron from the Fe-CAShexadecyltrimethylammonium bromide (HDTMA) complex, its color turns from blue to orange. The 1 ml of
DKC2 cell free supernatant was added with few drops of Fecl3 solution and OD was taken at different
wavelength by using spectrophotometer. It was found that supernatant showing a peak at 495 nm.
Fig. 1: Zone formation by strain DKM5 and DKC2 on CAS plates.
Effect of PGPR Strains on Seedling Growth of Chickpea:
The three strains DKM3, DKM5 and DKM9 showed stunning effect on root growth at 8 and 15 days in
chickpea as shown in fig 2. Maximum stunting effect on root was shown by strain DKM3 and DKM5 at 8 and 15
days followed by strain DKM9. A very slight stimulation on root was shown by strain DKC2 & DKM8. All the
strain was not showing any stimulation or stunting effect on shoot growth at both 5 and 15 day of seedling.
Co Inoculation of PGPR Strains with Mesorhizobium sp. cicer Ca 181:
Seed inoculation with Mesorhizobium sp. cicer Ca 181 alone or on coinoculation with PGPR strains (DKC2,
DKM3, DKM5, DKM8, and DKM9) increase the plant dry weight of chickpea in comparison to uninoculated
control as shown in fig 3. The plants dry weight increases by 2.06 times when inoculated with Mesorhizobium
and the plants dry weight increases from 2.06 to 3.22, 3.539, 3.74, 3.84 and 3.98 times when seeds were
184 Aust. J. Basic & Appl. Sci., 6(9): 183-187, 2012
coinoculated with DKM5, DKM3, DKM9 DKM8, and DKC2 respectively at 90 days and from 1.89 to 3.11, 3.30,
3.48, 3.6 and 3.75 times at 120 days as compare to control. The maximum gain in plants dry weight 3.98 and
3.75 times was found when seeds were coinoculated with DKC2 strain and Mesorhizobium at 90 and 120 days
respectively.
(A)
(B)
Fig. 2: Effect of PGPR strains on seedling growth (cm) (A- root, B- shoot) under aseptic conditions.
Fig. 3: Effect of co inoculation of PGPR strains with Mesorhizobium on plant growth.
Discussion:
Several studies have demonstrated the production of siderophore, other secondary metabolites and lytic
enzyme production by rhizospheric bacteria were involved in the control mechanism against plant root
pathogens (Nagraj kumar et al., 2004). Siderophore producing bacteria are good candidates for plant growth
promotion, especially in neutral to alkaline soil. In our study two PGPR strains DKC2 and DKM5 were found to
produce siderophore on CAS agar plates. The nature of siderophore produced by strain DKC2 was catecholate in
nature. The three PGPR strains (DKM3, DKM5, & DKM9) showed stunning effect on root growth at 8 and 15
days in chickpea, maximum stunting effect on root was shown by strain DKM3 and DKM5 at 8 and 15 days
followed by strain DKM9. A very slight stimulation effect on both root & shoot was shown by strain DKC2 &
DKM8. The root proliferation could be due to the production of antibiotic or siderophore like compound, the
stunting effect could be due to the contact of bacterial cells with legume seeds or due to synthesis or secretion of
excessive amount of IAA or some inhibitory agent when the bacterium was grown in synthetic medium or in
root exudates of chickpea. It is also possible that phytoalexins produced by seedlings as a host defense response
after inoculation of PGPR could be inhibitory for seedling growth. Similar improvement of seed germination
parameters by rhizobacteria has been reported in other cereals such as sorghum (Raju et al., 1999), and pearl
millet (Niranjan et al., 2004). The improvement in seed germination by PGPR was also found in work with
wheat and sunflower (Shaukat et al., 2006). It was found that some PGPR induced increases in seed emergence,
in some cases achieving increases up to 100 % greater than controls. These findings may be due to the increased
synthesis of hormones like gibberellins, which would have triggered the activity of specific enzymes that
promoted early germination, such as amylase, which have brought an increase in availability of starch
assimilation.
In this study, it was found that seed with Mesorhizobium sp. cicer Ca 181 alone or on co inoculation DKC2,
DKM3, DKM5, DKM8 and DKM9 increase the plant dry weight of chickpea in comparison to uninoculated
control as shown in Fig 3. The maximum gain in plants dry weight 3.98 and 3.75 times was found when seeds
were coinoculated with DKC2 strain and Mesorhizobium at 90 and 120 days respectively. The results are
supporting the other studies on coinoculation of PGPR with rhizobia on plant growth. Coinoculation
of Pseudomonas sp. with rhizobia has been reported to enhance nodulation, plant dry matter and grain yield in
other legumes like clover (Derylo and Skorupska, 1993), pea (Bolton et al., 1990) and soybean (Dashti et al.,
185 Aust. J. Basic & Appl. Sci., 6(9): 183-187, 2012
1998). These results suggested that PGPR strains (DKC2, DKM3, DKM5, DKM8, and DKM9) acted
synergistically with the Mesorhizobium sp. cicer strain Ca181 and were effective in promoting growth of
chickpea. The bacterial strain DKC2 can be used for dual purpose, for plant growth promotion and chlorpyrifos
degradation in soil. Since bacterium showing its pesticide degradation and plant growth promoting activity more
characterization of this bacterial strain is needed for its commercialization.
ACKNOWLEDGEMENT
We thank the Director, UIET, Kurukshetra University, for their great support. We also, thank faculty of
Dept. of Biotechnology, UIET for their support in finishing the project. I am also thankful to Dr. Rakesh
Sharawat, Assistant professor, Department of microbiology, CCSHAU, Hisar, Haryana, India to provide the
culture of Mesorizobium for this study.
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