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9
CHAPTER 2
REVIEW OF LITERATURE
The rhizosphere is the region around the root and has high nutrient availability. This is
due to the loss of as much as 40% of plant photosynthates from the roots (Lynch, 1991).
As a result, this region contains large and active microbial populations that may exert
beneficial, neutral, or detrimental effects on the growth of plant. The rhizospheric
microbial populations play a major role in maintaining root health, nutrient uptake, and
environmental stress tolerance (Bowen and Rovira, 1999 and Cook, 2002). Such micro-
organisms are important components of management practices so as to achieve the crop
yield. The crop yield refers to the attainable yield that is limited only by the natural
physical environment of the crop and its innate genetic potential (Cook, 2002).
The growth of plants in agricultural soils is influenced by various biotic and abiotic
factors. Many different physical and chemical approaches have been used by the growers
for the management of soil environment for the improvement of crop yields. But
application of microbial products for this purpose is a less common practise. Although,
rhizobial inoculants has been used to ensure efficient nitrogen fixation for legumes in
North America for over 100 years (Smith, 1997). The idea of manipulating rhizospheric
microbial populations of crop by inoculating beneficial soil bacteria for enhancement of
plant growth has proven to be successful in laboratory and greenhouse studies, but
responses have been quite distinct in the field (Bowen and Rovira, 1999). Major benefits
of this approach include reduced use of agricultural chemicals along with sustainable
management practices. With increasing understanding of the biological interactions
occuring in the rhizosphere, it is important to consider factors helpful in increasing the
technology’s reliability in the field thereby facilitating its commercial development
(Nelson, 2004).
Enhanced plant growth by bacteria has been reported by various researchers
throughout the world (Cooper, 1959; Mishustin and Naumova, 1962; Brown, 1974;
Kloepper et al., 1980a and Schippers et al., 1995). With better understanding of
rhizosphere and different mechanisms of action of PGPR, practical aspects of inoculant
formulation and delivery increases. This may lead to development of newer PGPR
10
products. Plant Growth Promoting Rhizobacteria (PGPR) are a group of bacteria that are
helpful in enhancement of plant growth and yield. These improve growth by different
plant growth promoting substances as well as biofertilizers. PGPR presents an
environmentally sustainable approach for enhancing crop production and health. With
the application of molecular tools, it is possible to manage the rhizosphere efficiently,
that may lead to new products with improved effectiveness.
As a consequence, a number of PGPR, e.g. Bacillus subtilis A13 (Turner and
Backman, 1991), B. licheniformis CECT5106 (Probanza et al., 2002), B. pumilus
CECT5105 (Probanza et al., 2002); Enterobacter cloacae UW4 and CAL2 (Shah et al.,
1998; Li et al., 2000 and Penrose and Glick, 2001), and others like P. fluorescens Pf-5,
P. fluorescens 2-79, P. fluorescens CHA0 (Wang et al., 2000), Pseudomonas putida
GR12-2 (Jacobson et al., 1994) etc. have been identified. Bacteria, especially
pseudomonads and bacilli have been found in the rhizosphere of various leguminous
crops. These bacteria effectively colonize the roots and suppress soilborne
phytopathogens (Parmar and Dadarwal, 2000).
The interactions between PGPR and rhizobia may be synergistic or antagonistic. The
beneficial effects of these interactions can be exploited for increasing the biological
nitrogen fixation and crop yield (Dubey, 1996). Plant growth-promoting Bacillus strains
have been reported to be present in the root nodules of soybean plants (Yu Ming et al.,
2002). Due to the harmful effects of artificial fertilizers on the environment and their
high cost, there has been increase in the use of beneficial soil microorganisms such as
PGPR for sustainable agriculture all around the world. PGPR are considered as efficient
biofertilizers for susutainable agriculture thereby improving crop yields.
11
2.1 OCIMUM SP. (TULSI)
Fig 1 Ocimum Plant
Ocimum (Tulsi), medicinal herb is considered as a sacred plant by the Hindus in
the Indian subcontinent. It also known as basil. Tulsi is described as sacred (Wealth of
India, 1991) and medicinal plant in ancient literature. This plant belongs to the family
Lamiaceae, characterized by square stem and specific aroma. Ocimum sanctum (Linn.) is
the most prominent species amongst the genera. O. sanctum, O. gratissimum, O.
basilicum, O. basilicum sub sp. minimum, O. americanum, O. kilimandscharium are
common species found in India. Among these, Ocimum sanctum (Linn.) is widely
distributed throughout the country from Andaman and Nicobar islands to the Himalayas
up to 1800 meters above the sea level (WOI, 1991). Ocimum sanctum has two varieties
i.e. black (Krishna Tulsi) and green (Rama Tulsi), which are chemically similar (Philip
and Damodaran, 1985) but both have common medicinal properties (Ghosh, 1995).
Several researchers have reported antioxidant, insecticidal, nematocidal, antifungal and
antibacterial activity of basil essential oils (Sangwan et al., 1990; Kelm and Nair, 1998;
Wan et al., 1998 and Griffin et al., 1999). Ocimum species produces characteristic
flavours and fragrances used in food, cosmetic and medicinal products. Tulsi improves
digestive system and possess properties such as anti- ulcer activity, anti-stress activity,
anti-carcinogenic, anti-oxidant, antimicrobial, anti-diabetic, anti-inflammatory and
protects heart and central nervous system (Table 1).
12
Due to the presence of essential or volatile oil, mainly concentrated in the leaves,
this plant has specific aromatic odor and contains phenols, terpenes and aldehydes. Fixed
oil, i.e. the oil, which is obtained from seed is mainly composed of fatty acids. Besides
oil, the plant also contains alkaloids, glycosides saponins and tannins. The leaves contain
ascorbic acid and carotene as well (WOI, 1991). The present day information about the
chemical properties is based on the various studies conducted in various countries.
However, the chemical constituents may vary due to edaphic and geographic factors. The
chemical constituents present in the plant reported in various literatures are shown in
table 2.
Table 1. Therapeutic uses of Tulsi
S.n
o.
Property and Plant
part used
Response Reference(s)
Antidiabetic
1. Ethanolic extract of O.
sanctum L.
Decreases the blood glucose,
glycosylated hemoglobin and urea with
a concomitant increase in glycogen,
hemoglobin and protein in
streptozotocin-induced diabetic rats
Narendhirak
annan et al.,
2006
2. O. sanctum L. leaf
extracts
Stimulatory effects on physiological
pathways of insulin secretion
Hannan et
al., 2006
3 O. sanctum L. extract for
30 days to normal rats
fed with fructose for 30
days
Lowered serum glucose level in
comparison with control group
Grover et al.,
2005
4 Possible mechanism of
glucose-lowering
activity of O. sanctum L.
in male mice.
Decreases the serum concentration of
both cortisol and glucose and also
exhibited antiperoxidative effect.
Therefore O. sanctum L. may
potentially regulate corticosteroid-
induced diabetic
Gholap and
Kar, 2004
5 Ethanolic extracts of
O. sanctum leaves
Partially attenuates sterptozotocin-
induced alterations in glycogen content
and carbohydrate metabolism in rats
Vats et al.,
2004
6 (O. sanctum L.) Leaf
powder
Significant reduction in fasting blood
sugar urogenic acid, total amino acids
level
Rai et al.,
1997
7 Alcoholic extract of
leaves of O. sanctum L.
Marked lowering of blood sugar level
in normal, glucose-fed hyperglycemic
and streptozotocin-induced diabetic
rats.
Chattopadhy
ay, 1993b
Cardiac activity
8 Hydroalcoholic extract Chronic-resistant stress/induced rise in Sood et al.,
13
of O. sanctum L. plasma cAMP level, myocardial
superoxide dismutase and catalase
activities as well as light microscopic
changes in myocardium in rats
2006
9 Leaf homogenate of O.
sanctum L.
Augments cardiac endogenous
antioxidants and prevents isoproterenol-
induced myocardial necrosis in rats
Sood et al.,
2005
10 Hydroalcoholic extract
of O. sanctum L.
Significantly reduced glutathione
(GSH), superoxide dismutase and LDH
levels.
Sharma et
al., 2001
11 Urosolic acid isolated
from O. sanctum L.
Protector against adriamycin induced
lipid peroxidation.
Balanehru
and
Nagarajan,
1992
Wound healing activity
12 Aqueous extract of O.
sanctum L.
Useful in the management of abnormal
healing such as keloids and hypertropic
scars in rats
Shetty et al.,
2006
13 Ethanolic extract of
leaves of O. sanctum L.
Significantly increased the wound
breaking strength, wound epithelializes
fast and wound contraction for normal
wound healing and dexamethasone-
depressed healing
Udupa et
al., 2006
Radioprotective effect
14 Aqueous extract of O.
sanctum L.
Significant reduction in lipid
peroxidation in kidney and salivary
glands in mice exposed to high doses
(3.7 MBq) of oral 131 iodine
Bhartiya et
al., 2006
15 Two polysaccharides
isolated from O. sanctum
L.
Prevent oxidative damage to liposomal
lipids and plasmid DNA induced by
various oxidants such as iron, AAPH
and gamma radiation
Subramanian
et al., 2005
16 Two water-soluble
flavonoids, Orientin and
Vicenin, isolated from
leaves
Significant protection against 4 Gy of
cobalt-60 gamma radiation, lethality
and chromosomal aberration in vivo
Vrinda and
Uma Devi,
2001 and
Hannan et
al., 2006
17 Aqueous extract of
leaves of O. sanctum
Increased the GSH and enzymes and
reduced the lipid peroxidation against
radiation lethality (Adult Swiss mice)
Devi and
Ganasoundar
i, 1999
18 Ocimum flavonoids
Orientin or Vicenin
Reduction in per cent aberrant cells
against 2 Gy gamma radiation (Adult
Swiss mice)
Devi et al.,
1998
19 leaf extract of O.
sanctum L. in
combination with WR-
2721 (WR) on mouse
bone marrow
Significant decrease in aberrant cells as
well as different types of aberrations ,
exposed to 4.5Gy gamma irradiation
(Adult Swiss mice)
Ganasoundar
i et al., 1998
14
Genotoxicity
20 O. sanctum L. aqueous
leaf extract against
chromium (Cr) and
mercury (Hg)-induced
genotoxicity
Significant recovery in mitotic index
(MI) and chromosomal aberrations (In
vivo cytogenetic assay in Allium cepa
root tip cells)
Babu and
Uma
Maheswari,
2006
21 Immu-21, a poly-herbal
formulation containing
O. sanctum L. and other
herbal extracts
Inhibited both cyclophosphamide (40
mg/kg)-induced classical and non-
classical chromosomal aberration (40–
60% of control).
Jena et al.,
2003
Antioxidant
22 Essential oils In hypoxanthine xanthine oxidase
assay, strong antioxidant capacity was
evident from O. sanctum L. (IC50 = 0.46
μl/ml).
Trevisan et
al., 2006
23 Aqueous extract of O.
sanctum L.
Significantly increases the activity of
anti-oxidant enzymes such as
superoxide dismutase, catalase level in
extract-treated group compared to
control.
Gupta et al.,
2006
24 Aqueous extract of O.
sanctum L.
Inhibit the hypercholesterolemia-
induced erythrocyte lipid peroxidation
activity in a dose-dependent manner in
male albino rabbits
Geetha and
Vasudevan,
2004
25 Methanolic extract of O.
sanctum L. leaves
Prevented reperfusion-induced rise in
lipid peroxidation and superoxide
dismutase. Also pretreatment also
stabilized the levels of tissue total
sulfhydryl group during reperfusion.
Yanpallewar
et al., 2004
Hypolipidemic
26 O. sanctum L. seed oil Significantly decreases serum
cholesterol, triacylglycerol and LDL +
VLDL cholesterol in cholesterol-fed
rabbits
Trevisan et
al., 2006
27 Fresh leaves of O.
sanctum L.
Significant lowering in serum total
cholesterol, triglyceride, phospholipids
and LDL-cholesterol level and
significant increase in the HDL-
cholesterol and total fecal sterol
contents of albino rabbits
Sarkar et al.,
1994
Antimicrobial
28 Linoleic acid in O.
sanctum L. fixed oil
Good antibacterial activity against
Staphylococcus aureus, Bacillus pumius
and Pseudomonas aeruginosa.
Singh et al.,
2005
29 Aqueous and alcoholic
extracts of O. sanctum L.
Activity against Klebsiella, E. coli,
Proteus, S. aureus and Candida albicans
when studied by agar diffusion method
Geeta et al.,
2001
15
Effect on gene transcription
30 Polyphenols extracted
from O. sanctum L.
Inhibits transcriptional expression of
genes (LDRL, LxRalpha, CD-36)
which control lipid metabolism,
cytotoxin production and cellular
activity within the arterial wall
Kaul et al.,
2005
Gastroprotective
31 Methanolic extract of
leaves of O. sanctum L.
Showed dose-dependent ulcer
protective effect against cold-restraint
stress-induced gastric ulcers
Goel et al.,
2005
32 Ocimum sanctum L. at a
dose of 100 mg/kg
Anti-ulcerogenic activity in cold-
restraint(CRU) (65.07%), aspirin (ASP)
(63.49%), alcohol (53.87%, pyloric
ligation (PL) (62.06%) induced gastric
ulcer models in rats, histamine-induced
(HST) (61.76%) duodenal ulcer in
guinea pigs and ulcer healing activity in
acetic acid induced chronic ulcer model
Dharmani et
al., 2004
Immunomodulatory effect
33 Aqueous extract of O.
sanctum L. leaf
Reduced the total bacterial count and
increased neutrophil and lymphocyte
counts with enhanced phagocytic
activity and phagocytic index in bovine
sub-clinical ma
stitis (SCM)
Mukherjee et
al., 2005
34 O. sanctum L. seed oil Modulate both humoral and cell-
mediated immune responsiveness in
both stressed and non stressed animals.
Mediratta et
al., 2002
35 Methanolic extract and
an aqueous suspension
of O. sanctum L. leaves
Immunostimulation of humoral
immunogenic response (Widal
agglutination and sheep erythrocyte
agglutination tests) as well as by
cellular immunologic response (E-
rosette formation and lymphocytosis) to
antigenic challenge of Salmonella
typhosa and sheep erythrocytes
Godhwani
et al., 1988
Sexually transmitted disease
36 Extract of O. sanctum L. Inhibition of Neisseria gonorrhoeae
clinical isolates and WHO organization
strains
Shokeen et
al., 2005
Effect on central nervous system (CNS)
37 Ethanol and chloroform
extractives of stem, leaf
and stem calli
Anticonvulsant, effective in preventing
tonic convulsions induced by
transcorneal electroshock
Jaggi et al.,
2003
38 Ethanolic extract of
leaves
Prolonged the time of lost reflex in
mice due to pentobarbital, decreased the
recovery time and severity of
electroshock and pentylenetetrazole-
induced convulsions and decreased
apomorphine -induced fighting time
Sakina et
al., 1990
16
and ambulation in ‘open field’ studies
39 Aqueous extract of
derived whole plant of
O. sanctum L.
Nootropic and anti-amensic agent in
mice; beneficial in the treatment of
cognitive disorders such as dementia
and Alzheimer's disease.
Joshi and
Parle, 2006
40 Methanolic extract of O.
sanctum L. root extract
Central nervous system stimulant
and/or anti-stress activity of O. sanctum
L.
Maity et al.,
2000
Antinociceptive (Analgesic)
41 Alcoholic leaf extract Reduced the number of writhes and
increased the tail withdrawal latency in
mice.
Khanna and
Bhatia, 2003
Anti-fertility activity
42 Benzene extract of O.
sanctum L. leaves
Reversible anti-fertility effect;
decreases the total sperm count, sperm
motility and forward velocity.
Ahmed et
al., 2002
Anthelmintic activity
43 Essential oil Eugenol, predominant component of the
essential oil exhibited an ED50
of 62.1
μg/ml; suggested as the putative
anthelmintic principle.
Asha et al.,
2001
Antiinflammatory
44 Compounds isolated
from O. sanctum L.
extract, Civsilineol,
Civsimavatine ,
Isothymonin, Apigenin,
Rosavinic acid and
Eugenol
Anti-inflammatory activity or
cyclooxygenase inhibitory activity:
Eugenol (97%), Civsilineol (37%),
Civsimavitin (50%), Isothymonin
(37%), Apigenin (65%) and Rosavinic
acid (58%).
Kelm et al.,
2000
45 Linoleic acid in fixed oil Blocking of cyclooxygenase and
lipoxygenase pathways of arachidonate
metabolism
Singh, 1998
46 Methanolic extract and
an aqueous suspension
of O. sanctum L.
Inhibited acute as well as chronic
inflammation in rats
Godhwani et
al., 1987
Anticancer
47 Fresh leaf paste
(topically) aqueous and
ethanolic extract (orally)
Chemopreventive activity against 7,12-
dimethylbenzaanthracene (DMBA)
induced (0.5%) hamster buccal pouch
carcinogenesis; increased the survival
rate and reducing the incidence of
papillomas and squamous cell
carcinomas
Karthikeyan
et al., 1999
48 Leaf extract Inhibiting metabolic activation of the
carcinogen
Prashar et
al., 1998
49 Seed oil Enhanced survival rate and delay in
tumor incidence in seed oil
Prakash and
Gupta, 2000
17
supplemented mice
Anticoagulant
50 Fixed oil Prolonged blood clotting time and the
response was comparable to that
obtained with aspirin (100 mg/kg).
Singh et al.,
2001
Anticataract
51 Aqueous Extract of fresh
leaves
Delayed the process of cataractogenesis
in experimental models (rats) of
cataract (galactosemic cataract in by
30% galactose and naphthalene cataract
in rabbits by 1 g/kg naphthalene)
Gupta et al.,
2005
Antiarthritic
52 Fixed oil Significantly reduced the diameter of
inflamed paw in formaldehyde-induced
arthritis in rats.
Singh et al.,
2007
Anti-plasmodial
53 Ethanolic leaf extract of
O. sanctum
Excellent antiplasmodial activity
(IC(50) 35.58 μg/mL) against
Plasmodium falciparum.
Inbaneson et
al., 2012
Toxicological properties
54 Ethanolic extract of Tulsi
Lethal dose in adult mice : LD50 of
Ocimum sanctum : 4505±80 mg/kg BW
on oral administration route and
3241±71 mg/kg BW by intra-peritoneal
routes
Bhargava
and Singh,
1981
55 Tulsi leaves aqueous and
alcoholic extracts
The acute LD 50 (30) values for
aqueous and alcoholic extracts were
found to be 6200 mg/kg BW and 4600
mg/kg BW respectively (mice).
Devi and
Ganasoundar
i, 1995
56 Fixed oil (seed oil) LD (50) of fixed oil: 42.5 ml/kg BW.
In acute toxicity study, no mortality at
30 ml/kg BW while 100% mortality
observed at 55 ml/kg BW.
Singh et al.,
1996
Antimicrobial properties
57 Oil Inhibitory effects on growth of
Mycobacterium tuberculosis and
Micrococcus pyogenes var. aureus. It
has one-tenth anti-tubercular potency
that of streptomycin and ¼ that of
isoniazid.
WOI, 1991
58 Aqueous and acetone
extracts of Ocimum
sanctum
sensitive to many plant fungi,
Alternaria tenuis, Helminthosporium
spp. and Curvularia penniseli
Sekhawat
and
Prasada,
1971
59 Essential oil Plant pathogenic fungi such as
Alternaria solani, Candida
guillermondii, Colletotricum capsici,
Curvularia spp., Fusarium solani,
Rao and
Nigam, 1970
and Dey and
Choudhuri,
18
Helminthosporium oryzae.
Bacterial organisms i.e. Anthrobacter
globiformis, Bacillus megaterium,
Escherichia coli, Pseudomonas spp.
Staphylococcus aureus, Staphylococcus
albus and Vibrio cholerae
1984
60 Essential oil Effective against both Gram-positive
bacteria and Gram-negative bacteria
and the properties were comparable
with the effectiveness of Clove oil
Prasad et al.,
1986 and
Phadke et
al., 1989
61 Aqueous, alcoholic
extract and seed oil of
Tulsi
Potential antimicrobial properties
against enteric pathogens
Geeta et al.,
2001 and
Singh et al.,
2005
62 Seed oil Effective against multi-drug resistant
Neisseria gonorrhoeae
Shokeen et
al., 2005 and
Shokeen et
al., 2008.
63 Ethanolic extracts Inhibitory effects on both clinical
isolates of β- lactamase producing
methicillin resistant Staphylococcus
aureus (MRSA) and methicillin -
sensitive Staphylococcus aureus
[MSSA]
Aqil et al.,
2005
64 Essential oil Activity against Propionibacterium
acnes and the minimum inhibitory
concentration (MIC) value found to be
3.0% v/v
Viyoch et
al., 2006
65 Leaves essential oil Fresh leaves essential oil : more
antibacterial properties compared to
dried leaves essential oil and in case of
fungus the property was just the reverse
Mondal et
al., 2005
Adaptogenic (Anti-stress) properties
66 Ethanolic extracts Prevented hepatotoxicity and
leukocytosis when administered in a
dose of 100 mg/kg body weight (BW).
stress tests includes: swimming
endurance test, milk induced
leukocytosis, aspirin induced ulcers and
carbon tetrachloride induced
hepatotoxicity
Bhargava
and Singh,
1981
67 Essential oil Reduced the LDH and alkaline
phosphatase levels due to restrain stress
in rats. Enhanced aspartate
transaminase and membrane dynamics
of RBC were reversed to near
normalcy.
Sen et al.,,
1992
68 Ethanolic extracts of
Tulsi leaves
Prevent the elevation in plasma
glucocorticoid levels following acute
Sebulingam
et al., 1997
19
and chronic noise stress in rats (100
mg/kg BW)
69 Ethanolic extract of the
roots of Ocimum
sanctum
Increased the mean swimming time
significantly when experimental mice
were subjected to swimming stress test
(400 mg/kg BW)
Maity et al.,
2003
70 Methanolic extract of
fresh leaves
Effective in bringing back to normal,
the altered values of acute noise
induced neutrophil functions
Archana and
Namasivaya
m, 2000.
71 A polyherbal
formulation containing
Tulsi along with other
plant extracts such as
Withania somnifera,
Tribulus territories and
Shilajeet
Treated animals showed reduction in
various induced stress related outcome
results and was comparable with the
proven adaptogen Ginseng
Bhattacharya
et al., 2000
72 Methanolic extracts of
Tulsi
Dose : 50/100 mg /kg BW significantly
reduce various paradigms of oxidative
stress caused by ischemia-reperfusion
injury, cigarette smoke, foot shock and
iron overload hepato-toxicity
Bhattacharya
et al., 2001
73 Fresh leaves Reduced the effects of hypoxia induced
oxidative damage
Sethi et
al.,2003
74 Alcoholic extract of
Tulsi
Inhibit lipid peroxidation of
erythrocytic membrane in a dose
dependant manner
Ravindran et
al., 2005
Miscellaneous properties
75 Tulsi extract Faster recovery of experimentally
induced wound in rats
Shetty et al.,
1991 and
Shetty et al.,
2006
76 Ocimum sanctum extract Treated human lymphocyte culture have
shown to reduce experimentally
induced genotoxic effects i.e.
chromosomal aberrations, mitotic
index, sister chromatid exchange and
replication index in a dose dependent
manner
Siddique et
al., 2007
77 Polyherbal formulation,
that also contained Tulsi
extract
Improved catalepsy score and super
oxide dismutase activity when
administered orally
Nair et al.,
2007
78 Tulsi extract (antioxidant
activities) of Ocimum
Sanctum
Preservation of packed food from
rancidity; can be used as a preservative
Juctachote
and
Berghoter,
2005
79 Ocimum sanctum L.
extract
Protection against HgCl2-induced
toxicity in mice
Sharma et
al., 2002
80 Aqueous extract of O.
sanctum L
Most effective aldose reductase
inhibitor (38.05%) inhibition of
Halder et al.,
2003
20
Table 2: Composition of chemical constituents of Tulsi (Ocimum sanctum Linn.)
Plant Part Composition Reference(s)
Essential
oil
from
Leaves
α-Thujene, Octane, Nonane, Benzene, (Z)-3-
hexanol, Ethyl 2-methyl butyrate, α-pinene, β-
pinene, Toluene, Citronellal, Camphene, Sabinene,
Dimethyl benzene, Myrecene, Ethyl benzene,
Limocene, 1:8-cineole, Cis-β-ocimene, Trans-β-
ocimene, p-cymene, Terpiniolene, Allo-ocimene,
Butyl-benzene, α-cubebene, γ-terpene, trans-
linalool oxide, Geraniol, α-copaene, β-bourbonene,
β-cubebene, Linalool, Eugenol, Methyl eugenol, β-
farnesene, β-elemene, (E)-cinnamyl acetate,
Isocaryophyllene, β-caryophyllene, Iso-eugenol, α-
guaiene, α-amorphene, α-humulene, γ-humulene,
4,11-selinadiene, α-terpeneol, Isoborneol, Borneol,
Germacrene-D, α-selinene, β-selinene,
Myrtenylformat, α-muurolene, δ-cadinene,
Cuparene, Calamenene, Geraneol, Nerolidol,
Caryophyllene oxide, Iedol, Humulene oxide, α-
guaiol, τ-cadinol, α-bisbolol, (EZ)-farnesol, Cis-
sesquisabinene hydrate, Elemol, Tetradecanal,
Selin-11-en-4-α-ol, 14-hydroxy-α-humulene
Lawrence, 1972;
Pareek et al.,
1980,82; Dey et al.,
1980,84; Asthana et
al.,1984; Phillip and
Damodaran, 1985;
Maheshwari et al.,
1987; Verma et al.,
1989; Gupta et al.,
1996; Bhattacharya
et al., 1996; Ravid et
al.,1997; Raju et al.,
1999; Kothari et al.,
2004 and Mondal et
al., 2005.
Alcoholic
extract of
leaves/
Aerial
parts
Ursolic acid, Apgenin, Luteolin, Apignin-7-O-
glucuronide, Luteolin-7-O-glucuronide, Isorientin,
Orientin, Molludistin, Stigmsterol, Triacontanol
ferulate, Vicenin-2, Vitexin, Isovitexin,
Aesculectin, Aesculin, Chlorgenic acid, Galuteolin,
Circineol, Gallic acid, Gallic acid methyl ester,
Gallic acid ethyl ester, Procatechuic acid, Vllinin
acid, 4-hydroxybenzoic acid, Vallinin, 4-
hydroxybenzoic acid, Caffiec acid, Chlorogenic
acid, Phenylpropane glucosides 1, Phenylpropane
glucosides 2, β-Stigmsterol, Urosolic acid
Nair and
Gunasekaran, 1982;
Nguyen et al., 1993;
Skaltsa et al., 1987,
99; Norr and Wanger
1992 and Sukari et
al., 1995.
Fixed oil
from Seeds
Palmitric acid, Stearic acid, Linolenic acid,
Linoleic acid, Oleic acid, Sitosterol, Dilinoleno-
linolins, Linoleno-dilinolin, Hexourenic acid
Nadkarni and
Patwardhan, 1952
and
Singh et al., 1995.
Mineral
content
(Per 100g)
Vit. C (83 mg), Carotene(2.5 mg), Ca (3.15%), P
(0.34%), Cr (2.9 µg), Cu (0.4 µg), Zn (0.15 µg), V
(0.54 µg), Fe (2.32 µg), Ni (0.73 µg), Insoluble
oxalate
WOI, 1991 and
Narendhirakannan et
al., 2005.
21
2.2 RHIZOSPHERE
The rhizosphere is populated by a wide range of microorganisms. The bacteria
colonizing this region are called rhizobacteria (Schroth and Hancock, 1982). The term
‘rhizobacteria’ implies a group of rhizosphere bacteria competent in colonizing the root
environment (Kloepper et al., 1991).The rhizosphere is the narrow zone of soil
specifically influenced by the root system (Dobbelaere et al., 2003 and Walker et al.,
2003). In comparison to the bulk soil, the rhizosphere is rich in nutrients. This happens
due to the accumulation of a various plant exudates, such as amino acids and sugars,
which provides rich source of energy and nutrients for bacteria. This situation is better
supported by the fact that the number of bacteria in the rhizospheric region of plants is
generally 10 to 100 times higher than that in the bulk soil (Weller and Thomashow, 1994
and Gray and Smith, 2005).
Plant roots play a major role in providing the mechanical support as well as
facilitate water and nutrient uptake. Besides these, plant roots secrete wide variety of
compounds that attract soil microbial communities. Such chemicals, called as root
exudates are secreted by roots into the soils helps in promoting the plant-microbe
interactions and inhibiting the growth of the competing plant species (Nardi et al., 2000).
These exudates may act as attractants or repellants and their composition is dependent
upon the physiological status and species of plants and microorganisms. The quality and
quantity of root exudates is influenced by microbial activity in the rhizosphere which
affects rooting patterns and the supply of available nutrients to plants. These exudates are
metabolized by microbes as C and N sources, and the resulting molecules are utilized by
the plants (Kang et al., 2010).
Indeed, carbon fluxes are critical determinants of rhizosphere function. Root
exudation contributes about 5-21% of photosynthetically fixed carbon transported to the
rhizosphere (Marschner, 1995). The rhizosphere (soil), the rhizoplane, and the root are
different components interacting in the rhizosphere, of which the rhizosphere is the zone
of soil influenced by roots through the release of substrates that affect microbial activity.
The rhizoplane can be defined as surface of the root to which the soil particles adheres
strongly. Micro-organisms such as endophytes also colonize the root tissues (Barea et al.,
22
2005). When the micro-organisms colonize the rhizoplane or root tissues, it is known as
root colonization, whereas rhizosphere colonization refers to the colonization of the
adjacent volume of soil under the influence of the root (Kloepper et al., 1991; Kloepper,
1994 and Barea et al., 2005). Thus, the rhizosphere can be defined as any volume of soil
specifically influenced by plant roots and/or in association with roots hairs, and plant-
produced materials (Dessaux et al., 2009).
2.3 PLANT GROWTH PROMOTING RHIZOBACTERIA
Based on the effects on plant growth, bacteria associated with the plants can be
classified into beneficial, deleterious and neutral groups (Dobbelaere et al., 2003).
Different bacterial genera are involved in a number of biotic activities of the soil
ecosystem, thus making it more dynamic in terms of nutrient availability and sustainable
agriculture. These are vital components of soils (Chandler et al., 2008 and Ahemad et
al., 2009). They help in the stimulation of plant growth through nutrient mobilization in
soils and production of different plant growth regulators rendering protection against
phytopathogens either by controlling or inhibiting them. This improves the structure of
soil and helps in bioremediation of polluted soils. They do so by sequestering toxic
heavy metal species and degradation of xenobiotic compounds such as pesticides (Braud
et al., 2009; Hayat et al., 2010; Rajkumar et al., 2010; Ahemad and Malik 2011 and
Ahemad, 2012). The rhizobacteria are more efficient in the transforming, mobilizing and
solubilizing nutrients and therefore, are the major driving forces for recycling of
nutrients present in the soil leading to increased fertility of soil (Hayat et al., 2010 and
Glick, 2012).
The rhizosphere, representing the thin layer of soil surrounding the roots of the
plant and the soil adhereing to the roots, supports large active groups of bacteria known
as plant growth promoting rhizobacteria. When a rhizobacteria is introducted as an
inoculant onto the plant, it confers a positive or benefial effect on the plant growth
(Kloepper and Schroth, 1978). Efficient root colonization, ability to survive and
compete, and plant growth promotion are few important characteristics to be recognized
as an effective plant growth promoting rhizobacteria (Kloepper, 1994). PGPR rapidly
colonize the rhizosphere and promotes suppression of soil borne pathogens at the root
surface and stimulate plant growth (Bloemberg and Lugtenberg, 2001). Among PGPR,
fluorescent Pseudomonas are considered to be the most promising group. They help in
23
the biocontrol of different plant diseases and produce secondary metabolites such as
antibiotics, volatile compound, phytohormones and siderophores. Their ability to
promote plant growth is mainly due to the production of antibiotics, indole acetic acid
and siderophores. The genera of PGPR include Acetobacter, Azospirillum, Azotobacter,
Bacillus, Burkholderia, Paenibacillus, Pseudomonas and few members of the
Enterobacteriaceae. An area of extensive research is the direct use of microorganisms
for plant growth promotion and plant pest management. The initial step in the
pathogenesis of soil borne microorganisms is the ability to colonize the rhizosphere and
is important for the microbial inoculants to be used as biofertilizers, phytostimulators,
biocontrol agents and bioremediators. Pseudomonas sp. is often used as model root
colonizing bacteria (Lugtenberg et al., 2001).
The rhizosphere is defined as the zone of soil in which the microflora is
influenced by the root (Hiltner, 1904). There is a wide range of mechanism through
which PGPR may exert their beneficial effect on plants for growth promotion and
biocontrol. Production of phytohormone by PGPR help in stimulation of the growth of
plant roots (Brown, 1972). Seed bacterization has been major area of research for the
establishment of beneficial bacteria on roots systems (Brown, 1974). There have been
numerous attempts for the characterization and quantification of microorganisms
inhabiting this zone, with techniques such as direct observation with light or electron
microscopy to study the rhizosphere effect.
Beneficial free-living soil bacteria are usually referred to as plant growth-
promoting rhizobacteria (Kloepper et al., 1989). PGPR possess various attributes such as
antibiotics, production of phytohormones, siderophores, fixation of atmospheric nitrogen
and phosphate solubilization (Glick and Ibid, 1995). Motile rhizobacteria possess better
ability for rhizospheric colonization than the non - motile organisms. This contributes
towards better rhizosphere activity and transformation of nutrients. One way for the
elimination of deleterious rhizobacteria from the rhizosphere is by niche exclusion that
may promote plant growth. Another mechanism for biocontrol by PGPR is induced
systemic resistance (ISR) that manipulates the physical and biochemical properties of
the host plant for controlling plant diseases.
24
PGPR applications at commercial level are successful due to the better
understanding of the microbial interactions, resulting in enhanced plant growth (Farzana
et al., 2009). PGPR, a group of beneficial plant bacteria, has been potentially useful for
stimulation for plant growth and improvement of crop yields. The technology has
extensively evolved to the level where we can now use them successfully in field
experiments (Saharan and Nehra, 2011). Increased growth and yields of radish, sugar
beet, potato and sweet potato have been reported.
PGPR affect plant growth either directly or indirectly. The indirect way for plant
growth promotion is when PGPR inhibits or prevent the deleterious effects of one or
more plant pathogens whereas direct promotion occurs when PGPR either by producing
phytohormones or facilitating nutrient uptake from the environment (Glick, 1995).
About 1 to 2% of bacteria contributes in plant growth promotion in the rhizosphere
(Antoun and Kloepper, 2001). PGPR colonizing in the rhizosphere and root surfaces are
independent of vegetal growth promotion (Gray and Smith, 2005). Among so many
different bacterial genera recognized as PGPR, Bacillus and Pseudomonas spp. are
predominant (Podile and Kishore, 2006). PGPR and their associations with plants are
exploited commercially for growth promotion in plants for achieving sustainable
agriculture and such associations have been studied in barley, canola, cucumber, wheat,
oat, peas, tomatoes and wheat (Gray and Smith, 2005 and Podile and Kishore, 2006).
Currently, there is a strong emphasis to explore biological approaches for
improvement of crop production following integrated plant nutrient management system.
Different symbiotic and non-symbiotic rhizobacteria are now being used worldwide as
bio-inoculants for plant growth promotion and development under various stresses like
heavy metals, insecticides, herbicides, and fungicides (Ahemad and Khan, 2010b; Wani
and Khan, 2010; Ahemad and Khan, 2011, 2011a,b,c,d and Ma et al., 2011a,b). Among
symbiotic PGPR are Rhizobium, Bradyrhizobium, Mesorhizobium whereas
Pseudomonas, Bacillus, Klebsiella, Azotobacter, Azospirillum, Azomonas are non-
symbiotic PGPR. Although, the mechanisms of PGPR are not yet fully identified, these
properties help in augmenting plant growth and development (Khan et al., 2009 and
Zaidi et al., 2009).
25
2.3.1 The Importance of Plant Growth Promoting Rhizobacteria
The term ‘rhizobacteria’ was introduced by Kloepper and Schroth (1978) to those
soil bacteria which were able to colonize the roots of plants and promoted their growth
by reducing the plant disease incidence. The term ‘PGPR’ was also given by Kloepper
and Schroth (1981) to describe beneficial soil bacteria that colonize the plant roots and
helps in enhancing their growth. PGPR are the part of rhizospheric biota that helps in the
growth of plants through various mechanisms either directly or indirectly. Cook (2002)
described plant growth promoting rhizobacteria that has innate genetic potential for the
management of agricultural practices.
Introduction of bacteria in soil has been implied for achieving proper plant
growth (Cooper, 1959; Brown, 1974; Kloepper et al., 1980a and Schippers et al., 1995).
The increased understanding of rhizosphere, mechanisms of PGPR and ease of
formulation of inoculants is leading to newer PGPR products with increased potential.
PGPR has an advantage of environmentally sustainable approach for increasing crop
production and health. A recent area of research is biotization where microorganisms can
be used as co-culture for the production of biomass and secondary metabolites. For
example, increased production of phenolics has been recorded, when Origanum vulgare
L. plantlets were co-inoculated with Pseudomonas sp. (Nowak, 1998). Bacteria,
especially pseudomonads and bacilli in the rhizospheric region of leguminous crops are
reported to help in root colonization by rhizobia and in reduction of soilborne plant
pathogens (Parmar and Dadarwal, 2000).
2.3.2 Isolation of PGPR Strains
The seed bacterization with rhizobacteria may aid in improving plant growth and
biological control of root pathogens. This concept came into existence from the works of
Burr et al. (1978) and Kloepper et al. (1980b). They reported the growth promotion
effects of Pseudomonas strains which were antagonistic to phytopathogens in vitro.
These studies led to the idea of modifying the rhizosphere microbiota with
microorganisms introduced with the planting material. Plant growth promoting
rhizobacteria are those bacteria which inhabit root and rhizospheric soil and are helpful
in increasing growth of the plant (Kloepper et al., 1989). Many strains of Bacillus are
known to have PGPR activity and they have been studied as model organisms for
acquiring a broad knowledge of the mechanisms involved. These bacteria are present in
26
the immediate vicinity of plant roots. Bacillus subtilis establishes steady contact with
higher plants thereby enhancing their growth. Considerable colonization was observed
when Bacillus licheniformis was inoculated on tomato and pepper. Further, the strain did
not require any alteration in the normal management in green houses and as such can be
used as a biofertilizer (Garcia et al., 2004). These strains are known to release various
metabolites that enhance the nutrients availability to the plants (Charest et al., 2005).
Pseudomonas and Bacillus confers plant growth enhancement and control of diseases
against phytopathogens. These two processes are complex and interrelated. This may
involve mechanisms such as synthesis of phytohormones like auxin and gibberellins,
induction of ACC deaminase, phosphate solubilization, etc. (Hamid and Ahmad, 2010).
Pseudomonas sp. is a common bacteria occurring in agricultural soils. This
species possess a number of growth promotion traits, thus making it a suitable candidate
as PGPR. Under field conditions, Pseudomonas strain resulted in enhancement of
legume yield (Johri, 2001). Fluorescent Pseudomonas are the most effective strains of
Pseudomonas and eminent efforts are going worldwide to exploit their potential for use
as bioinoculants/biofertilizers. They help in maintaining soil health and are diverse in
terms of metabolism and function (Lata et al., 2002). Pseudomonas fluorescence
inoculant stimulated chickpea growth and yield (Rokhzadi et al., 2008). Considerable
enhancement in dry and fresh masses was recorded with the isolates of fluorescent
Pseudomonas obtained from rhizosphere soil of sugarcane (Mehnaz et al., 2009).
Few strains of fluorescent Pseudomonas can be applied as seed inoculants on
crops for promotion of growth and enhancement in yields. These are capable of rapid
and stable colonization with plant roots of potato, sugar beet and radish. They could
significantly increase yield upto 44% in field tests. Different environmental and plant
related factors may affect the occurrence and activity of microorganisms in the soil.
These include soil type, nutrient abundance, pH, moisture content, species, age, etc. This
can be exemplified in a case where among the two winter wheat cultivars,
Pseudomonas showed higher counts depending on the development phase of wheat
plants (Wachowska et al., 2006).
Pseudomonas spp. possessing PGP traits may be used as biofertilizers. They help
in increasing crop yield by various mechanisms either directly or indirectly (Walsh et al.,
27
2001). Several Pseudomonas strains have the ability for phosphorous solubilization
thereby increasing the availability of phosphorus to plants (Sundara et al., 2002).
Fluorescent Pseudomonas have been found in abundance in rhizosphere of various
crops (Kumar et al., 2004). Several Pseudomonas strains produce siderophores that
increase plant growth by increasing iron solubility in the rhizosphere of plants.
Siderophores are chelating agents that possess high affinity for absorption of iron. About
30 PGPR strains belonging to fluorescent Pseudomonas with PGP activities were
isolated from the rhizosphere of rice and characterized by PCR-RAPD analysis (Reddy
and Reddy, 2009).
About 32 bacterial isolates belonging to Pseudomonas putida, Pseudomonas
fluorescens and Serratia sp. were isolated from soil colonized soya been roots (Chanway
et al., 1989). Ramette et al. (2006) showed that isolated strains of Pseudomonas have
multiple PGP traits such as greater ability of plant hormone production, phosphate
solubilization and siderophore production. Sen et al. (2006) observed considerable
inhibition of growth of Sclerotium rolfsii by Pseudomonas BRL-1.
Egamberdieva (2010) analyzed the plant growth promoting bacteria for their
growth-stimulating effects on two wheat cultivars carried out in pot experiments using
calcarous soil. Bacterial strains Pseudomonas sp. and P. fluorescens colonized the
rhizosphere of both wheat cultivars and significantly stimulated the shoot and root length
and dry weight of wheat. About 144 rhizospheric bacteria were isolated from cucumber
and screened for their biocontrol activity against Phytophthora drechsleri, causal agent of
cucumber root rot by Maleki et al. (2010). Based on dual culture assays, eight isolates
were selected for root colonization and PGPR traits in greenhouse studies. Isolate CV6
showed the highest root colonization and significant plant growth promotion under in
vitro condition.
Deshwal et al. (2013) isolated 140 PGPR strains of Pseudomonas from potatoes
rhizosphere at Dehradun Valley, India. Malleswari and Bagyanarayana (2013) isolated
219 PGPR strains from the rhizosphere soil samples of different medicinal and aromatic
plants viz., Artemisia vulgaris, Acorus calamus, Coleus forskohlii, Ocimum sanctum,
Andrographis paniculata, Mentha spicata, Aloe vera, Tagetes erecta, Mimosa pudica
and Withania somnifera. In the rhizosphere of all medicinal and aromatic plants,
28
microbial population was high and these bacterial strains possessed multiple PGP
activities. Kannahi and Kowsalya (2013) isolated Pseudomonas fluorescens and Bacillus
subtilis and studied their effect on growth and development of Vigna mungo. Saranraj et
al. (2013) collected the paddy rhizosphere soil sample from ten different locations in
Cuddalore district of Tamil Nadu. The microbial population (bacteria, fungi and
actinomycetes) in the rhizosphere soil sample was estimated by serial dilution and pour
plating method. Pseudomonas fluorescens was isolated and characterized by gram
staining, motility test, plating on King’s B medium and bio-chemical tests. The
population of P. fluorescens ranged between 7.71 × 106 cfu/g and 7.21 × 10
6 cfu/g of
soil. Sixteen (16) putative of endophytic bacteria were isolated from sodicity tolerant
polyembryonic mango root stock of GPL-3 and ML-4 from Lucknow, India. Isolate
CSR-M-16 showed increased root and shoot length of rice followed by CSR-M-8, CSR-
M-9 and CSR-M-6 (Kannan et al., 2014).
2.3.3 Screening and Selection of PGPR Strains
To confer effective control of diseases to a particular plant, it has been proposed
that microorganism isolated from rhizosphere of that plant would be more adapted to the
crop than any other micro-organism from some different plant. These crop-associated
microorganisms would be better biocontrol agents due to their close association with the
plant. These are already adopted to the plant or to the prevailing environmental
conditions under which they are supposed to function. Such locally adopted strains
yielded improved biocontrol strains in some cases (Cook and Baker, 1983). Due to
microbial biodiversity studies, it is now feasible to identify potential bioagents that may
function successfully under different environmental conditions. Identifiying a potential
antagonistic strains is just an initial step for developing an effective biological control
agent. But to imply this on a commercial and larger level, the selected strain must be
ecologically fit to survive, become established and function within the particular
conditions of the ecosystem. After identification of potent strains, next step is to screen
these strains for specific mechanisms, interactions, conditions and requirements
responsible for effective biological control. With better understanding of the beneficial
characteristics and limitations of these strains, it would become possible to develop
strategies for their management and implementation. Use of markers helps in identifiying
biocontrol agents. Such a protocol offers successful development and commercialization
of technology for potential biocontrol agents.
29
2.3.4 Mechanisms of PGPR
PGPR are able to directly enhance plant growth by mechanisms such as
atmospheric nitrogen fixation that is transferred to the plant (termed Biofertilizers),
siderophore production (termed antifungal activity), solubilization of minerals such as
phosphorus, and phytohormones synthesis like auxins, cytokinins and ethylene synthesis
(termed Biostimulants), synthesis of anti-fungal metabolites (termed Bioprotectants) or
induction of systemic resistance (Kloepper, 1993; Glick, 1995; Frankenberger and
Arshad, 1995; Bloemberg, 2001; Persello-Cartieaux et al., 2003 and Nelson, 2004).
PGPR strains may use one or more direct or indirect mechanisms in the
rhizosphere. Few PGPR strains, when inoculated on the seed before planting may
establish themselves on the roots of the crop which a very common way for reduction of
damping-off (Pythium ultimum) among crops. Bacteria in the genera Bacillus,
Streptomyces, Pseudomonas, Burkholderia and Agrobacterium are the biological control
agents predominantly studied (Kloepper, 1993). Commercialized PGPR organism,
Bacillus subtilis has biocontrol potential against variety of pathogenic fungi (Boland and
Kuykendall, 1998). Direct enhancement of mineral uptake due to increase in specific ion
fluxes at the root surface in the presence of PGPR has also been reported (Bashan, and
Levanony, 1991 and Bertrand et al., 2000). Competition for an ecological niche or a
substrate, production of inhibitory allelochemicals and inducing systemic resistance
(ISR) in host plants are various mechanisms of bio-control mediated by PGPR for many
different phytopathogens (Bloemberg, 2001; Lugtenberg et al., 2001; Wang et al., 2001
and Compant et al., 2005).
Plant growth promoting rhizobacteria are beneficial bacteria present in the soil
and these bacteria may facilitate plant growth and development both directly and
indirectly (Glick, 1995). Direct stimulation may include sequestering of iron by bacterial
siderophores and soluble phosphate providing plants with fixed nitrogen,
phytohormones such as auxins, gibberellins, cytokinins and ethylene (Tien et al., 1979;
Scher and Baker, 1982; Loper, 1986; Arshad and Frankenberger, 1991; Mordukhova et
al., 1991; Boddey and Dobereiner, 1995; Glick et al., 1995; Gutierrez Manero et al.,
1996; de Freitas et al., 1997; Kennedy et al., 1997; Timmusk, 1999; Gutierrez Manero et
al., 2001 and Patten and Glick, 2002); while indirect promotion of plant growth includes
prevention of phytopathogens (biocontrol) (Glick and Bashan, 1997).
30
Antagonism against plant pathogens by β-1,3-glucanase, chitinases, antibiotics,
cyanide (Voisard et al., 1989; Renwick et al., 1991; Shanahan et al., 1992; Fridlender et
al., 1993 and Flaishman et al., 1996) and production of ACC deaminase for reducing
ethylene levels in the plant roots are few more mechanisms for increasing plant growth
(Jacobson et al., 1994; Glick, 1995; Glick et al., 1998; Li et al., 2000 and Penrose and
Glick, 2001). Terpenes, jasmonates, and green leaf components are various plant
volatiles that may act as potent signal molecules for plants and organisms of other
trophic levels (Farmer, 2001 and Farag, 2006). It has been reported that PGPR released
volatile components increased growth of Arabidopsis thaliana (Kloepper et al., 2003).
Bacillus subtilis (BSCBE4), Pseudomonas chlororaphis (PA23), endophytic P.
fluorescens (ENPF1) have been reported to inhibit mycelial growth of Corynespora
casiicola, causal agent of stem blight under in vitro (Mathiyazhagan et al., 2004).
2.3.5 Production of Plant Growth Promoting Substances by PGPR Isolates
2.3.5.1 Biological nitrogen fixation
Plants play a major role in selecting different types of bacteria by the constituents
of their root exudates. The nature and concentrations of organic constituents of exudates
and the corresponding ability of the bacteria to utilize these as energy source are the
major factors that decide the rhizosphere bacterial community structure. The
communities of bacteria present in the rhizosphere promote the uptake and catabolism of
organic compounds that are present in the root exudates quite efficiently (Barraquiro et
al., 2000). There are number of beneficial bacterial species associated with the plant
rhizosphere enhancing plant growth. Major beneficial bacterial genera include
Acinetobacter, Bacillus, Burkholderia, Enterobacter, Pseudomonas, Rhizobium and
Serratia (Tilak et al., 2005). Many of these bacteria attach themselves to plant root
surfaces and derive maximum benefit from the various compounds of root exudates.
Understanding the plant-microorganism interactions would be helpful to attain the use of
microorganisms as bioinoculants for sustainable agricultural system. Plant growth
promoting bacteria may be employed as inoculants for improving the growth and yield of
agricultural crops. The use of nitrogen fixing bacteria and beneficial microorganism as
potential biofertilizer/bioenhancer may aid in the reduction of chemical fertilizer
applications due to which overall production cost gets reduced. Apart from increasing the
productivity, PGPR also helps in reducing pollution and are eco-friendly in comparison
31
to the organic fertilizers (Stefan et al., 2008). Also, they seem to be an attractive
alternative for chemical pesticides/fertilizers (Ashrafuzzaman et al., 2009).
Nitrogen (N) is the principal plant nutrient. It is the most important nutrient in
terms of plant growth and productivity. Major portion of nitrogen (78%) is present in
unavailable form in the atmosphere. Leaching of minerals and losses due to rains might
be the reasoms for the limited availability of this vital nutrient. Biological nitrogen
fixation or diazotrophy is the conversion of atmospheric N to NH3 by nitrogen fixing
microorganisms using nitrogenase enzyme system (Kim and Rees, 1994). It provides a
low cost, eco-friendly alternate for chemical fertilizers (Ladha et al., 1997). The process
usually takes place at mild temperatures. The nitrogen fixing microorganisms are widely
distributed in nature (Raymond et al., 2004). The ability to fix nitrogen is widespread
among prokaryotes with representatives in both bacteria and archaea (Dekas et al.,
2009).
Increased biological nitrogen fixation was recorded due to the increased nodule
occupancy in soya bean with the combined inoculations of Bradyrhizobium sp. with
Pseudomonas striata (Dubey, 1996). Fluorescent pseudomonads were reported to
promote nodulation in chickpea, by increasing nitrogen fixation (Parmar and Dadarwal,
2000). Several PGPR strains are capable of fixing nitrogen and making them available to
plants for growth promotion. Few examples of efficient nitrogen fixing bacteria are
Azoarcus sp., Beijerinckia sp., Klebsiella pneumoniae, Pantoea agglomerans and
Rhizobium sp. (Antoun et al., 1998 and Riggs et al., 2001). They do so either
symbiotically (Azotobacter spp., Bacillus spp., Beijerinckia spp.) or non-symbiotically
(free living diazotrophics such as Azoarcus, Azospirillum, Burkholderia,
Gluconacetobacter and Pseudomonas) (Reinhold-Hurek et al., 1993; Dobereiner, 1997;
Estrada de los Santos et al., 2001; Barea et al., 2005; Mirza et al., 2006 and Bashan and
de-Bashan 2010).
Diazotrophs are PGPR that fix nitrogen in non-leguminous plants. They form a
non-obligate interaction with the host plants. Nitrogen fixation is carried out by
nitrogenase enzyme system. Dean and Jacobson (1992) elucidated the structure of
nitrogenase. It is a metalloenzyme consisting of two components. First is the
dinitrogenase reductase (iron protein) and second component is dinitrogenase having a
32
metal cofactor. The function of dinitrogenase reductase is to provide electrons that
possess high reduction power. Dinitrogenase utilizes these electrons for the reduction of
nitrogen to ammonia. These may differ in metal co-factors and classified into three
nitrogen fixing systems such as Mo-nitrogenase, V-nitrogenase and Fe-nitrogenase, with
most of the fixation being contributed by molybdenum nitrogenase present in all
diazaotrophs (Bishop and Jorerger, 1990). Nitrogen-fixing systems have different
structure in different bacterial genera (Kim and Rees, 1994 and Glick et al., 1999).
The nif genes are responsible for nitrogen fixation, occurs in symbiotic as well as
free living systems (Kim and Rees, 1994). In case of Rhizobium, symbiotic activation of
these genes depends on low concentration of oxygen. This is regulated by fixfix-genes,
common in symbiotic and free living nitrogen fixing bacteria (Dean and Jacobson, 1992
and Kim and Rees, 1994).
Seed treatment with PGPR resulted in increased yield and growth in potato under
field conditions (Kloepper et al., 1980a). Van Peer and Schippers (1988) documented the
increased root and shoot fresh weight of potato, cucumber, tomato and lettuce as a result
of bacterization with Pseudomonas strains. Among various biofertilizers, nitrogen fixing
and phosphorus solubilizing bacteria can be a potential PGPR in the biofertilization of
crops (Karlidag et al., 2007). These bacteria improve plant nutrition by increasing
nitrogen and phosphate uptake by plants. PGPR strains may enhance the plant growth
either by fixation of atmospheric nitrogen or by solubilization of minerals such as
phosphorus (Karthikeyan et al., 2007; 2008). Also, other mechanisms include production
of plant growth regulators for growth promotion (Klopper and Schroth, 1978 and Jaleel
et al., 2007).
PGPR strains are known to stimulate growth and yield in Ashwagandha and other
medicinal plants (Attia and Saad, 2001 and Thosar et al., 2005). This PGPR activity is
documented in species belonging to Acinetobacter, Azotobacter, Bacillus, Beijernckia,
Burkholderia, Enterobacter, Flavobacterium, Pseudomonas, Rhizobium and Serratia
(Rodriguez and Fraga, 1999; Sudhakar et al., 2000 and Karlidag et al., 2007). The
occurrence of Azotobacter, Azospirillum and Pseudomonas in the rhizosphere of
medicinal plants like C. roseus, Coleus forskholi, Ocmium sanctum and Aloe vera has
been also reported (Karthikeyan et al., 2008).
33
2.3.5.1.1 Increase in growth
Plant weight of tuber-treated potatoes increased by 80% on average by midseason and
emergence increases of 10-40% resulted for canola when seeds were coated with PGPR
before planting (Kloepper, 1981a; 1991). Yield increases between 10% and 20% with
PGPR applications have been documented for several agricultural crops (Kloepper,
1991).
Algawadi and Gaur (1992) reported combined inoculation of sorghum with A.
brasilense and phosphate solubilization bacteria; P. striata or B. polymyxa significantly
increased grain yield and dry matter content, N and P uptake as compared with single
inoculation of individual organisms. The stimulatory effects of this PGPR strains on the
yield and growth of these crops were attributed to the ability to fix nitrogen,
phytohormone synthesis and mineral solubilization (Kevinvessey, 2003; Cakmakci et al.,
2007 and Karlidag et al., 2007). For C. roseus, P. fluorescens is known to enhance
biomass yield and ajmalicine alkaloid content under water deficient stress (Jaleel et al.,
2007). The higher N, P and K content in PGPR combination treatment may have resulted
from the nitrogen fixation and P-solubilizing ability of these strains (Attia and Saad,
2001; Aslantas et al., 2007; Cakmakci et al., 2007 and Karlidag et al., 2007). The role of
PGPR strains in increase of the plant nutrient elements has been discussed (Sundara et
al., 2002; Shen et al., 2004 and Karlidag et al., 2007). The effect of plant growth
promoting rhizobacteria (PGPR) inoculation on microbial community structure in
rhizosphere of forage corn cultivated in Thailand was studied. It was reported that forage
corn seeds inoculated with Pseudomonas sp. SUT 19 and Brevibacillus sp. SUT 47
mixed with compost promoted biomass and growth of forage corn (Piromyou et al.,
2011).
A study was carried out to study the effects of inoculation with Glomus
fasciculatum and PGPR namely Bacillus megaterium and Pseudomonas fluorescens on
growth and biomass of Ocimum basilicum under glass house conditions. Single and dual
inoculations increased the growth and biomass, compared to uninoculated plants. The
consortium of all the three organisms was found superior in enhancing plant height,
yield, and P content. Mycorrhizal colonization and spore numbers in the root zone soil
were significantly increased in G. fasciculatum inoculated treatment and its combination
with PGPR (Hemavathi et al., 2006). Pandey et al. (2014a) investigated the effect of
34
Glomus fasciculatum and fluorescent Pseudomonas on growth and productivity of
Ocimum sanctum in pot culture to assess the impact of these two on biomass yield.
Pseudomonas fluorescens and Glomus fasciculatum when applied in pot trials
considerably increased the growth as well as yield of Ocimum sanctum applied with Zn
amended soil.
The inoculation of Pseudomonas fluorescens and Glomus fasciculatum in
Ocimum sanctum enhanced seed germination over control under pot trial studies.
Inoculation of Pseudomonas fluorescens and Glomus fasciculatum enhanced all the
growth parameters of plant under pot trials. Pandey et al. (2014b) studied the impact of
various physico- chemical parameters of soil on the growth of Ocimum sanctum under
various treatments. The application of Pseudomonas fluorescens and Glomus
fasciculatum in pot trials substantially increased the growth and yield of Ocimum
sanctum.
2.3.5.1.2 Increase in root length and dry weight
PGPR are able to exert a beneficial effect upon plant growth such as increase in root
growth and root weight. In addition, it is known that growth promotion and increase in
root formation in response to PGPR inoculation may involve various mechanisms. There
have been reports regarding induction of root formation in stem cuttings by bacteria
belonging to genera Agrobacterium, Bacillus, Streptomyces, Pseudomonas and
Alcaligenes (Bassil et al., 1991; Rinallo et al., 1999 and Ercisli et al., 2004).
Xia et al. (1990) reported that PGPR inoculation can enhance plant dry weight.
Hall et al. (1996) mentioned that when the canola, lettuce, tomato, and wheat seeds were
treated with P. putida GR12-2, average root length increased. Yan et al. (2003) reported
that the population of PGPR strains Bacillus pumilus and P. fluorescens colonizing
tomato roots after application into the soil less medium showed higher population on the
whole roots and lateral roots than on the tap roots. Ribaudo et al. (2006) reported that
inoculation with A.brasilense FT 326 increased root fresh weight of tomato plants.
Inoculation of wheat with Azospirillum brasilense wild strains increased root hair
formation. A mutant of Azospirillum brasilense with production of phytohormones, but
with high nitrogenase activity did not enhance root over uninoculated controls. Increased
root growth was reported in C. roseus under treatment with PGPR (Karthikeyan, 2008).
35
Kaymak et al. (2008) reported effect of some bacteria isolates on length and dry matter
content of mint roots. Agrobacterium rubi A16, Bacillus subtilus OSU142, Bacillus
megatorium M3, Burkholderia gladii BA7 and Pseudomonas putidea BA8 were used as
rooting agents.
2.3.5.1.3 Increase in plant height, alkaloid and nutrient content
Gopal (2004) revealed that the inoculation of PGPR increased the plant height, number
of leaves, number of laterals and root diameter and increased fresh and dry weight and
seed yield in Ashwagandha. Considerable enhancement in emergence ability and weight
of seedling has been reported by IAA producing Pseudomonas strains (PGPR) (Han and
Lee, 2005). Jaleel et al. (2008) investigated the effect of plant growth regulators and
fungicide treatments on the growth characteristics of Catharanthus roseus. They found
an increase in plant height in C. roseus under treatment with PGPR P. fluorescens.
Similar results were reported in C. roseus under different PGPRtreatments. P.
fluorescens increased plant height in C. roseus. PGPR produced high quantities of
extracellular indole acetic acid (IAA) and tryptophol in culture medium supplemented
with tryptophan, a precursor of IAA. Karthikeyan et al. (2009) discussed the effect of
different plant growth promoting rhizobacteria (PGPR) like Azospirillum brasilense and
Pseudomonas fluorescens on growth parameters and the production of terpenoid indole
alkaloids are investigated in two varieties ‘rosea’ and ‘alba’ of Catharanthus roseus.
Karthikeyan et al. (2010) noted increased alkaloid content, root length and girth
of roots, and plant height of Catharanthus roseus by PGPR (Azotobacter, Bacillus and
Pseudomonas). In The results of study suggest that PGPR applied in combination have
the potential to increase the alkaloid level, growth and nutrient content of C. roseus.
Ordookhani (2011) investigated the antioxidant activity of essential oil and
microelements of Ocimum basilicum with PGPR such as P. putida 41, A. chroococcum
5, and A. lipoferum OF and found that the microelement contents and antioxidant activity
of essential oil were increased by PGPR treatments in comparison to the control
treatment. The maximum antioxidant activity of essential oil and Fe, Mn and Cu contents
were obtained in Pseudomonas putida strain 41+ Azotobacter chroococcum strain +
Azosprillum lipoferum strain OF treatment and maximum of Zn content found in
Azotobacter chroococcum strain + Azosprillum lipoferum strain OF treatment.
36
2.3.5.2 Indole Acetic Acid
Plant hormones can be defined as chemical messengers that influence the plant’s ability
to respond to its environment. These are organic compounds synthesized in one part of
the plant for transport to another location and are quite effective at very low
concentration. Also called as plant growth regulators, due to their ability to stimulate or
inhibit plant growth. Five major groups of hormones are: auxins, gibberellins, ethylene,
cytokinins and abscisicacid.
IAA (auxin) is a member of phytohormones. This hormone is known to influence
a number of cellular functions in plants, hence, considered as important regulators of
plant growth and development. It has been proved that IAA synthesis occurs in many
plant-associated bacteria through which they can aid in plant growth promotion (Patten
and Glick, 1996; 2002). IAA is considered to be the most important native auxin
(Ashrafuzzaman et al., 2009) and functions as an important signal molecule in the
regulation of plant development. General functions of IAA includes plant cell division,
extension, and differentiation; stimulation of seed and tuber germination; increasing the
rate of xylem and root development; initiation of lateral and adventitious root formation;
affects photosynthesis, formation of pigment, biosynthesis of various metabolites, and
resistance to stressful conditions (Glick, 2012).
IAA plays a major role in root initiation, cell division and cell enlargement
(Salisbury, 1994). IAA is important for plant growth and development. The diversity of
function is explained due to the complexity of IAA biosynthetic, transport and signaling
pathways (Santner et al., 2009). It also acts as a reciprocal signaling molecule that affects
the expression of various genes in many microorganisms. It is plays a vital role in
rhizobacteria-plant interactions (Spaepen and Vanderleyden, 2011).
About 80% of rhizospheric microorganisms isolated from different crops are able
to synthesize auxins as secondary metabolites (Patten and Glick, 1996). It has been
reported that due to the secretion of IAA by rhizobacteria, the endogenous pool of plant
IAA may be altered and this leads to disturbances in the ongoing plant developmental
processes (Spaepen et al., 2007 and Glick, 2012). Synthesis of IAA occurs through
different pathways that are widespread among plant-associated bacteria.
37
Although IAA produced by rhizobacteria disturbes the plant auxin pool, but, it
increases the length and surface area of plant roots by providing increased accessibility
to plants for the nutrients present in the soil. Thus, rhizobacterial IAA is identified as an
effector molecule in plant-microbe interactions, both in pathogenesis and
phytostimulation (Spaepen and Vanderleyden, 2011). Also, rhizobacterial IAA tend to
loosen the plant cell walls that causes greater root exudation providing more and more
nutrients and supports the growth of rhizospheric bacteria (Glick, 2012).
Tryptophan (amino acid) has the ability to alter IAA synthesis levels. It acts as a
precursor for IAA and stimulates IAA production. On the other hand, anthranilate is a
precursor for tryptophan but causes reduction in IAA synthesis. The mechanism is quite
well balanced in a way that tryptophan causes inhibition of formation of anthranilate by a
negative feedback regulation on the anthranilate synthase. This leads to indirect
induction of IAA production (Spaepen et al., 2007 and Zaidi et al., 2009). There have
been numerous reports suggesting higher IAA levels produced by rhizobacterial strains
in culture medium amended with tryptophan (Spaepen and Vanderleyden, 2011).
Acidic pH, osmotic and matrix stress, and carbon limitation are factors present in
the environment that may exert stress in bacteria and causes modulation in their IAA
biosynthesis (Spaepen et al., 2007). Since, IAA and nodule formation seems to be related
processes. IAA influences formation of vascular bundle, division and differentiation
ability of cells, important for nodule formation. When IAA biosynthetic pathway was
introduced in Rhizobium leguminosarum bv. viciae, it was observed that root nodules
formed contained 60-fold more IAA as compared to the nodules formed by the wild-type
Rhizobium in Vicia hirsute (Camerini et al., 2008). The auxin level seems to be
necessary for nodule formation in the leguminous plants (Spaepen et al., 2007 and Glick,
2012). IAA production is reported in most of the Rhizobium species (Ahemad and Khan,
2011; 2011b; 2012b, c, e).
Different factors such as plant’s sensitivity to IAA, amount of bacterial IAA
produced, and other phytohormone production also effect IAA levels (Peck and Kende,
1995). IAA is commonly produced by PGPR (Barazani and Friedman, 1999). Bacterial
IAA produced by P. putida greatly influenced the development root system in the host
plant (Patten and Glick, 2002. It was reported that P. fluorescens HP 72 produced IAA
38
helped in the suppression of creeping bentgrass brown patch (Suzuki and Oyaizu, 2003).
The effects of IAA production have been implicated in the plant growth promotion by
PGPR (Vessey, 2003). IAA producing strains stimulates plant growth. When such strains
are inoculated into the crop, considerable increases were recorded in the plant growth of
sweet potato cultivar by the N, K, Ca and Mg uptake (Farzana and Radizah, 2005).
Considerable enhancement was observed in rooting and root dry matterof cuttings of
eucalyptus in substrate inoculated with IAA producing rhizobacteria. Few rhizobacterial
strains stimulate the rhizogenesis and plant growth. This has led to significant increase in
the yield of rooted cuttings in clonal nurseries (Teixeria et al., 2007). IAA producing
PGPR strains increased the plant growth significantly, when inoculated to cucumber,
tomato and pepper plants (Kidoglu et al., 2007). Independent of the origin (rhizosphere
vs. phyllosphere), IAA producing bacterial strains synergistically enhanced the growth of
peas and wheat. The highest IAA levels were recorded by P. fluorescens and Kocuria
varians (Ahmad et al., 2005 and Egamberdieva, 2008).
Various phytohormones produced by PGPR influence plant growth and
development. Studies suggest that low auxins concentration stimulates plant growth
whereas high concentrations may be inhibitory to the plant (Arshad and Frankenberger,
1991). A wide variety of soil microflora has the ability to synthesize auxins in pure
culture and soil (Barazani and Friedman, 1999). The auxin biosynthesis by rhizobacteria
can be used as a tool to select more effective PGPR strains (Khalid et al., 2004).
Maximum increase in growth and yield of the wheat crop was achieved with strains that
produced the highest levels of auxins (indole acetic acid (IAA) and indole acetamide
(IAM) in non-sterilized soil (Khalid et al., 2004). Different plant seedlings respond
differently to variable auxin concentrations and type of microorganisms (Sarwar and
Frankenberger, 1994 and Ahmad et al., 2005). It has been reported that the strains
producing lower IAA levels, releases it continuously (Tsavkelova et al., 2007).
According to Spaepen et al. (2007), IAA can act as a signaling molecule in bacteria and
directly influences bacterial physiology.
There have been reports where microorganisms produce IAA in the presence of a
suitable precursor such as L-tryptophan. The tryptophan increased IAA production in
Bacillus amyloliquefaciens. Azospirillum produced IAA when exposed to tryptophan
(Tien et al., 1979).
39
It was found that colonization of strain HP72 on the bent grass root induced root growth
reduction. In case of HP72LI, no growth reduction was observed. Also, strain HP72
showed higher colonization ability on the bentgrass root than HP72LI. IAA production
with fluorescent Pseudomonas isolates was evaluated in pure culture in the absence and
presence of L-tryptophan (Karnwal, 2009). It was observed that, with increase in the
concentration of tryptophan, indole production increased. Cassana et al. (2009) has
reported that Azospirillum brasilense strain Az39 and Brayrhizobium japonicum strain
E109 excreted IAA into the culture medium and produced morphological and
physiological changes in young seed tissues of Corn (Zea mays L) and Soybean (Glycine
max L) promoting their early growth. PGPR isolates promoted growth of rice by
inducing IAA production (Ashrafuzzaman et al., 2009). Plants inoculated with the
rhizobia together with Ag+ ion and L-tryptophan (Trp), produced the highest root dry
weight, and considerably enhanced the uptake of phosphorus, nitrogen and potassium
(Etesami et al., 2009).
IAA producing isolates stimulated the growth in plants by enhanced nutrient (N,
P, K, Ca, Mg) uptake in sweetpotato cultivar (Farzana and Radizah, 2005). Bacillus
megaterium isolated from tea rhizosphere was found to produce high levels of IAA
promoting plant growth (Chakraborty et al., 2006). IAA production induced increased
root and shoot weight of wheat and bacterial survival (Narula et al., 2006). IAA-
mediated production of ethylene by PGPR inoculation resulted in increased number of
root hair, biomass and surface area of tomato plant roots (Ribaudo et al., 2006). Few
phosphate-solubilizing bacteria and fungi are able to produce IAA and act as plant
growth promoters. PSB and PSF differ in their potential for IAA production (Souchie et
al., 2007). IAA producing Burkholderia sp. MSSP and Sinorhizobium meliloti PP3
increased seedling growth in Cajanus cajan (Pandey and Maheshwari, 2007). According
to Swain et al. (2007), Bacillus subtilis strains capable of producing IAA showed
beneficial effect in Dioscorea rotundata growth.
In a study conducted on isolates obtained from paddy rhizosphere, P. fluorescens
isolates were screened for IAA and siderophore production. Isolate PF-8 exhibited
maximum IAA production while minimum IAA level was recorded by the isolate PF-4
(Saranraj et al., 2013). IAA biosynthesis was studied in P. fluorescens Psd to assess the
40
growth promotion potential of this strain (Sivasakthivelan et al., 2013). Due to the lack
of indole pyruvic acid (IPyA) pathway, the indole acetamide (IAM) pathway(observed
in phytopathogens)was expressed in strain Psd. Due to the overexpression of IAM
pathway genes iaaM-iaaH (from Pseudomonas syringae subsp. Savastanoi), IAA levels
increased drastically leading to a negative effect on root development in sorghum.
2.3.5.3 Phosphate Solubilization
The improvement of soil fertility is one of the most common strategies to
increase agricultural production. Most importantly, biological N fixation plays a vital
role in enhancing the soil fertility. Phosphorus (P), second only to nitrogen nutrient in
requirement for plants, is major essential macronutrients for biological growth and
development. Major portion of phosphorous in soil exists as non-utilizable insoluble
phosphates that plants cannot take up directly (Pradhan and Sukla, 2006).
Microorganisms may solubilize insoluble inorganic P of soil making it available to the
plants. This ability of some microorganisms is quite important for yield enhancement in
plants (Chen et al., 2006 and Rodriguez et al., 2006). Such rhizobacterial strains may act
as efficient growth promotion agents in agricultural crops (Chaiharn et al., 2008).
There has been much research interest for improving plant growth by bacteria
capable of solubilizing mineral phosphates leading to increased P availability. Enhanced
phosphate availability to rice has been attributed to the PGPR’s ability to solubilize
precipitated phosphates, promoting plant growth under field condition (Verma et al.,
2001). Enhanced phosphorus uptake by plants is reported by the use of PSB as
inoculants (Igual et al., 2001 and Chen et al., 2006). Phosphate Solubilising
Microorganisms (PSM) including bacteria has provided an alternative biotechnological
solution in sustainable agriculture to meet the P demands of plants. The most efficient
phosphate solubilizers among bacteria belong to genera Bacillus, Rhizobium and
Pseudomonas. Among fungi, Aspergillus and Penicillium are known to be efficient
phosphate solubilizers. It is now possible to manage our agriculture system in a more
sustainable way due to increased knowledge regarding the mechanisms, colonilizing
abilities and commercial applications of such beneficial microorganisms (Zaidi et al.,
2009).
41
Phosphorus plays role in numerous plant processes including generation of
energy, photosynthesis, respiration and nucleic acid synthesis, Plants can absorb
phosphorus only as H2PO4- and HPO4
2- ions. Although, soil contains sufficient amounts
of phosphate to support plant growth, most of the organic and inorganic forms are
inaccessible to the plant. Phosphorus is widely applied as a chemical fertilizer and upon
reaching the soil can be fixed into insoluble forms that are inaccessible to plants
(Rodriguez and Fraga, 1999; Igual et al., 2001; Vance et al., 2003 and Smyth, 2011).
There are different ways by which plants react to limitation of phosphorus. These include
acidification of the rhizosphere, increased growth of roots towards unexploited soil
zones, increased number of root hairs and secretion of phosphatases. Secretion of organic
anions and protons, with citrate and oxalate being most effective causes acidification,
facilitating phosphate mobilisation (Richardson et al., 2009). Eutrophication and hypoxia
of lakes and marine estuaries are few detrimental effects of excessive and unmanaged
phosphorus application (Smyth, 2011).
Phosphate-solubilising bacteria possess the ability to solubilise bound phosphorus
from organic or inorganic molecules and makes it available to the plant and are
ubiquitous (Lipton et al., 1987; Kim et al., 1998; Igual et al., 2001 and Gyaneshwar et
al., 2002). Potent phosphate solubilizing species include Bacillus, Enterobacter, Erwinia
and Pseudomonas spp. Mesorhizobium ciceri and Mesorhizobium mediterraneum are
two chickpea nodulating species reported to be efficient phosphate solubilizers (Rivas et
al., 2006). According to Richardson et al. (2009), mechanisms such as acidification,
organic acid production, proton secretion, chelation and exchange reactions are reported
to be involved in the conversion of inorganic forms of P to the utilizable form in various
PSM.
Microbial processes such as production of organic acid and proton extrusion
leads to phosphate solubilization (Nahas, 1996). Phosphate starvation might also cause
solubilization of phosphate (Gyaneshwar et al., 1999). Phosphate solubilization is known
to take place via chelation-mediated mechanisms in saprophytic bacteria and fungi
(Whitelaw, 2000). In soil, phosphorus concentration gets altered by root exudates such as
organic ligands (Hinsinger, 2001). Most microorganisms, through their metabolic
activities exudate organic acids. Such acids dissolves rock phosphate, or chelate calcium
ions and releases phosphorus into the solution. Another reason for generation of
42
phosphate forms is release of a wide range of enzymes such as non-specific
phosphatases, phytases and phosphonatases and C-P lyases (Idriss et al., 2002 and
Rodriguez et al., 2006). Release of phosphorus from mineral phosphate is majorly
contributed by gluconic acid production (Rodriguez et al., 2006). It is to be known that
phosphate solubilization and mineralization can coexist in the same bacterial strain (Tao
et al., 2008). Among the phosphate solubilizing microorganisms prevailing in the
rhizosphere, PSB may act as promising biofertilizers due their ability to supply
phosphorus to plants from sources otherwise scarcely available by various mechanisms
(Zaidi et al., 2009).
Rhizobacteria have been reported to promote growth in a large number of
agricultural crops like potato, tomato, wheat, radish, pulses etc. Few examples of
phosphate solubilizers intimately associated with these crops are Azotobacter
chroococcum, Bacillus circulans, Bradyrhizobium japonicum, Cladosporium herbarum,
P. putida, Enterobacter agglomerans, P. chlororaphis and Rhizobium leguminosarum
(Antoun et al., 1998; Chabot et al., 1998; Kim et al., 1998; Cattelan et al., 1999; Kumar
and Narula, 1999 and Singh and Kapoor, 1999). Among the most significant phosphate
solubilizing bacterial genera are Azospirillum, Azotobacter, Bacillus, Beijerinckia,
Enterobacter, Erwinia, Microbacterium, Pseudomonas, Rhizobium and Serratia (Sturz
and Nowak, 2000 and Mehnaz and Lazarovits, 2006).
Significant increase in the yield of canola by phosphate-solubilising Bacillus spp.
was recorded (de Freitas et al., 1997). Researchers suggest that availability of
phosphorus limits the process of nodule formation. Although a major portion of the
supplemented phosphorus reacts with soil components becoming non-utilizable, legumes
like alfalfa and clover displayed a high positive response to phosphorus supplementation
(Gyaneshwar et al., 2002). The genus Bacillus accounts for nearly 95% of Gram-positive
soil bacilli (Garbeva et al., 2003). These are endospore forming capable of surviving
under adverse conditions. Few species are diazotrophs such as Bacillus subtilis, while
many others possess multiple PGPR traits (Timmusk et al., 1999; Probanza et al., 2002;
Kokalis-Burelle et al., 2002; Garcia et al., 2004 and Barriuso et al., 2008).
Enhance nutrient uptake (phosphorus) by AM fungi has been reported (Bodker et
al., 1998). Significantly release of phosphorus was reported by Rhizobium
43
leguminosarum bv. viciae due to extracellular oxidation of glucose to gluconic acid via
the quinoprotein glucose dehydrogenase (Goldstein and Rogers, 1999). Phosphate
solubilizing isolates obtained from soyabean rhizosphere increased soyabean growth
significantly (Cattelan et al., 1999). Phosphate-solubilizing Bacillus megaterium was
found toincrease sugarcane yield and available phosphorus content for the plant (Sundara
et al., 2002). Azotobacter vinelandii and Bacillus cereus were found to solubilize
phosphate in vitro. They may promote plant growth (Husen, 2003).
About 4800 bacterial isolates were obtained from the root-free soil, rhizospheric
region and the rhizoplane region of P. juliflora occurring in alkaline soils. Highest
number of PSB was found in the rhizoplane region followed by rhizosphere soil (Tilak et
al., 2005). It has been reported that Bacillus M3 alone or in combination with Bacillus
OSU-142 significantly enhanced the yield, growth and nutrition of raspberry plant under
organic growing conditions (Orhan et al., 2006). Bacillus megaterium isolated from tea
rhizosphere was found to be phosphate solubilizing thereby enhancing the plant growth
(Chakraborty et al., 2006). In an investigation by Ramachandran et al. (2007), significant
phosphate solubilization was observed with Pseudomonas sp. and Azospirillum sp.
obtained from the rhizosphere soil and root cuttings of Piper nigrum.
Significant increase in growth and phosphorus content of maize was reported by
phosphate-solubilising Pseudomonas sp. (Vyas and Gulatti, 2009). Phosphate
solubilizing E. coli obtainedfrom endorhizosphere of sugarcane (Saccharum sp.) and rye
grass (Lolium perenne) promoted growth in plants (Gangwar and Kaur, 2009).
Identification and characterization of soil PSB for the effective plant growth-promotion
broadens the spectrum of phosphate solubilizers available for field application.
PSB are common inhabitants found in most soils. Environmental stress disturbes
their establishment and performances (Ahemad and Khan, 2010; 2010a; 2012a,d). There
have been several reports regarding the positive effects of PSB inoculation used either
alone or in combination with other rhizospheric bacteria (Zaidi and Khan, 2005; Chen et
al., 2008; Poonguzhali et al., 2008; Vikram and Hamzehzarghani, 2008; Ahemad and
Khan, 2010b and Ahemad and Khan, 2012c). PSB stimulates the efficiency of BNF
contributing to plant growth. They also increase the availability of other trace elements
through production of PGP compounds (Suman et al., 2001; Ahmad et al., 2008 and
44
Zaidi et al., 2009). Recently, agricultural microbiologists are showing keen interest in
PGPRs with phosphate solubilizing potential due to their contribution in effective plant
growth.
2.3.5.4 Hydrogen Cyanide Production
The cyanide ion is exhaled as HCN and metabolized to a lesser degree in to other
compounds. HCN acts as a general metabolic inhibitor, by inhibiting electron transport
which disrupts the energy supply to the cell causing death of the organisms. Cyanide has
toxic properties. It is synthesized, excreted and metabolized by many organisms, such as
bacteria, algae, fungi, plants, insects, etc. as a mean to avoid predation or competition. It
inhibits proper functioning of enzymes and natural receptors reversible mechanism of
inhibition (Corbett, 1974). Glycine is known to be a carbon precursor for HCN in P.
aeruginosa (Castric, 1977). It differs from cyanogenesis in other bacteria due to two
reasons. Firstly, all other amino acids except glycine cause stimulation of HCN
production and secondly, both carbons of glycine are used as sources of cyanide carbon.
HCN is a commonly produced secondary metabolite by pseudomonads present in the
rhizosphere. It imparts negative effects on root metabolism and root growth (Schippers et
al., 1990) and inhibits the action of cytochrome oxidase (Gehring et al., 1993). It seems
to be an environment friendly means for weed biocontrol (Heydari et al., 2008).
For positive regulation of HCN biosynthesis, low oxygen concentrations are
essential for the activity of the transcription factor ANR (Pessi and Haas, 2000).
Increased levels of supplemental glycine led to increased HCN production in root-free
soil by P. putida and A. delafieldii, with P. putida typically generating 8-38 μM of HCN
at a given glycine level (Owen and Zdor, 2001). Cyanide-producing bacteria as
inoculants do not impart any negative effect on the host plants. Host-specific
rhizobacteria may be considered as efficient biological weed-control agents (Zeller et al.,
2007). Studies suggest that HCN production is a common trait of Pseudomonas
(88.89%) and Bacillus (50%) in the rhizospheric soil and plant root nodules (Charest et
al., 2005 and Ahmad et al., 2008). HCN is found to be a biocontrol metabolite in
Pseudomonas species.
Several rhizobacteria are known to produce HCN involved in biological control
of pathogens. The induction and alteration of plant physiological activities by the
45
cyanide producing strain CHA0 has been reported to stimulate root hair formation
(Voisard et al., 1989). HCN production by various fluorescent pseudomonads strains are
reported for suppression of soil borne pathogens (Voisard et al., 1989 and Defago et al.,
1990). HCN production was found to suppress black root rot of tobacco and take-all of
wheat by P. fluorescens strain CHA0 (Stutz et al., 1986 and Defago et al., 1990). The
mycelial growth of Pythium was inhibited by the HCN producing Pseudomonas
fluorescens under in vitro conditions (Weststeijn, 1990). HCN producing PGPR strains
were reported to induce systemic resistance in cucumber against Colletotrichum
orbiculare (Wei et al., 1991). Seed germination and root length was found to increase
significantly by the HCN producing strain of fluorescent Pseudomonas RRS1 obtained
from Rajanigandha (tuberose) (Saxena et al., 1996). HCN from P. fluorescens strain
CHA0 not repressed by fusaric acid played a significant role in control of disease caused
by pathogenic fungi F. oxysporum f. sp. radicis-lycopersici in tomato (Duffy et al.,
2003). HCN, a broad spectrum antimicrobial compound produced by fluorescent
pseudomonads associated with plants is found to be effective in biocontrol of root
disease (Ramette et al., 2003). It was noted that HCN synthase (enzyme) is encoded by
three biosynthetic genes (henA, henB and henC).
Most of the rhizosphere isolates assessed for HCN production in vitro, produced
HCN and promoted plant growth (Wani et al., 2007). Chickpea rhizosphere isolates
produced HCN that promoted plant growth directly or indirectly or synergistically along
with other PGP traits (Joseph et al., 2007). HCN producing Mesorhizobium loti MP6, a
rhizosphere competent strain was found to enhance the growth of Brassica campestris
under normal growth conditions (Chandra et al., 2007). No significant change was
reported in HCN production abilities of Bacillus and Pseudomonas isolates obtained
from mustard rhizosphere, on application of herbicides such as quizalafop-p-ethyl and
clodinafop (Munees and Mohammad, 2009). Seed bacterization with a psychrotolerant,
HCN producing Pseudomonas fragi CS11RH1 (MTCC 8984) considerably increased
percent germination, germination rate, plant biomass and nutrient uptake of wheat
seedlings (Selvakumar et al., 2009). Also, HCN production is reported by an
entomopathogenic bacterium Pseudomonas entomophila (Ryall et al., 2009).
46
2.3.5.5 Siderophore Production
One of the necessary elements for growth of all living forms is iron and an abundant
element on the earth crust. Due to the limited availability of iron in soil and plants,
intense competition is generated (Loper and Henkels, 1997). Due to the difficulty in
solubility, it is not easily accessible for uptake by the organism. The ferric ion (Fe3+
)
available is only 10-18 M. This is the most available form of iron accessible to
organisms and gets binded to chelators produced by plants. In the roots, the ferric ion is
reduced to the ferrous ion (Fe2+
) and taken up by the plant. Also, the plant can absorb
iron ion as a Fe3+
-phytosiderophore complex (Lemanceau et al., 2009).
Siderophores in Greek noun refers to "iron carrier". These are small, high -
affinity iron chelating compounds that are secreted by organisms like bacteria, fungi and
grasses (Miller and Marvin, 2008). These siderophores have low molecular weight, less
than10kD and synthesized by microbes in high amounts under iron limiting conditions.
In the aerobic environment at physiological pH, unavailability of iron is due to its
occurrence as Fe3+
that readily forms insoluble hydroxides and oxyhydroxides. Plants
and microorganisms are unable to uptake iron in this insoluble form (Rajkumar et al.,
2010).
Siderophores tend to possess high specific activity for chelation of ferric ions.
They act as vehiclesforthetransportof ferric ironinside the microbial cell. They possess
different types of ligands. These ligands can be hydroxamate, catechol or carboxylate
(Hofte, 1993). Some bacteria are able to produce a variety of siderophores under iron
limited conditions. These siderophores may bind ferric ions with high affinity. Initially
the ferric-siderophore complex is formed that is recognized by specific membrane
receptors and is transported actively through membranes of Gram-negative and Gram-
positive bacteria (Boukhalfa and Crumbliss, 2002).
Siderophores can be referred as small peptidic molecules containing side chains
and functional groups that furnish a high-affinity set of ligands for formation of
coordination complexes with ferric ions available in the environment (Crosa and Walsh,
2002). Siderophore producting PGPR may generate furious competition for root
colonization and exclusion of other microorganism from their ecological niche (Haas and
Defago, 2005). For instance, during intense competition for the available carbon sources
47
due to secretion of exudates by roots depends on the ability of absorbing iron via
siderophores (Crowley, 2006). On the basis of structural features, ligand-types and
functional groups that form coordination complex with iron, four classes of bacterial
siderophores have been recognized: carboxylate, hydroxamates, phenol catecholates and
pyoverdines (Crowley, 2006).
Siderophores are able to chelate ferric ion with high affinity enabling its
solubilization and extraction from most mineral or organic complexes (Wandersman and
Delepelaire, 2004). In aerobic conditions, due to the unstable nature of ferrous (Fe2+
)
form, it is readily oxidized to the oxidized ferric (Fe3+
) form. These oxidized forms exist
as hardly soluble ferric hydroxide that is inaccessible to biological systems (Krewulak
and Vogel, 2008 and Osorio et al., 2008). Although several plant species can absorb
Fe3+
-siderophore complexes formed by bacteria, still it is not yet confirmed that uptake
of such complexes really benefits in promoting plant growth (Zhang et al., 2008).
Numerous studies have been conducted on siderophore production and identification, for
their use in enhancement of plant health. Several of them are widely recognized and
commonly produced by a large variety of microorganisms. Although, studies suggests
that few of them are also species-specific (Crowley, 2006 and Sandy and Butler, 2009).
This mechanism of enhancing plant health and growth is found to be crucial for
identifying efficient PGPR in soil. Microbial siderophores are helpful in increasing the
uptake of iron by plants capable of recognizing bacterial ferric-siderophore complex. It
has been reported that production of siderophore resulted in enhanced iron uptake in
presence of metals like nickel and cadmium (Burd et al., 1998 and Dimkpa et al., 2008;
2009). Whether such bacterial siderophores are able to meet the iron nutrition demands
of the plants is still uncertain.
Siderophores produced by pseudomonads are known to possess the highest
affinity for iron among all bacterial siderophores studied so far. The more potent
pyoverdin was reported to inhibit the less potent siderophore producing bacteria and
fungi in vitro in an iron depleted media (Kloepper et al. 1980a). Suppression of
Fusarium oxysporum by pseudobactin (siderophore) produced by P. putida B10 strain
has been observed in soil deficient in iron. It was found that on replenishing the soil with
iron, the suppression was lost. This happened because replenishment of soil with iron
causes repression of siderophore production in microorganisms (Kloepper et al., 1980b).
48
Several studies have shown that siderophores produced by fluorescent pseudomonads
suppressed many soil-borne fungal pathogens by their ability to form complexes with the
available iron so that it is no longer available to other organisms for uptake (Kloepper et
al., 1980a and Neilands, 1995).
Siderophores are also important for some pathogenic bacteria for their acquisition
of iron. Enterobactin is one of the strongest siderophores that binds tightly to ferric ions
(Raymond et al., 2003). Many of these are non-ribosomal peptides but few get
biosynthesized independently (Neilands, 1952, 1995; Challis, 2005; Miethke and
Marahiel, 2007 and Miller and Marvin, 2008). In a study based on distribution of
amplified ribosomal DNA restriction analysis (ARDRA), it was found that most of the
siderophore producing isolates belong to Gram negative bacterial genera such as
Pseudomonas and Enterobacter and Gram positive bacterial genera such as Bacillus and
Rhodococcus (Tian et al., 2009). Most of the siderophores are water-soluble and can be
divided into extracellular siderophores and intracellular siderophores. There is a marked
difference between the siderophore cross-utilizing ability of rhizobacteria. Some
rhizobacteria can proficiently utilize the siderophores produced by members the same
genus (homologous siderophores) where as others can use only those produced by other
rhizobacteria of different genera (heterologous siderophores) (Khan et al., 2009). After
the reduction of ferric ion to ferrous ion, release of iron ions in the cell is carried out by a
tripping mechanism, which connects the inner membrane of the outer membranes.
During this reduction process, the siderophore may be destroyed / recycled. This
mechanism is similar in both Gram-negative and Gram-positive rhizobacteria (Neilands,
1995 and Rajkumar et al., 2010). Other than iron, siderophores are reported to form
stable complexes with heavy metals including Cd, Cu, Ga, Pb, Zn, and radionuclides (U
and Np). Thease heavy metals and radionuclides are of significant environmental
concern (Kiss and Farkas, 1998; Neubauer et al., 2000 and Indiragandhi et al., 2008).
When a siderophore binds to a metal, it increases the solubility of that metal thereby
increasing its availability levels (Rajkumar et al., 2010). By doing so, bacterial
siderophores helps the plants in relieving stress imparted by the presence of heavy metals
in soils.
Direct uptake of siderophore-Fe complexes or by a ligand exchange reaction or
chelation and release of iron from bacterial siderophores are few different mechanisms
49
through which iron assimilation occurs in plants (Schmidt, 1999). In an investigation,
siderophore-production by Pseudomonas strain GRP3 was assessed for its beneficial
effects on iron nutrition of Vigna radiate (Sharma et al., 2003). Increased levels of iron,
chlorophyll a and chlorophyll b were observed as well as chlorotic symptoms declined
considerably. A siderophore mediated- iron transport system was reported in oat plants
and was further studied (Crowley and Kraemer, 2007). The oat plant was found to
possess mechanisms for utilizing iron-siderophore complexes formed under iron-limited
conditions by various siderophore producing- rhizosphere microorganisms. The iron-
pyoverdine complex synthesized by Pseudomonas fluorescens C7 was reported to
facilitate iron uptake by Arabidopsis thaliana plants, resulting in increased iron content
in plant tissues and improved plant growth (Vansuyt et al., 2007). There have been
several reports confirming enhanced plant growth via siderophore-mediated Fe-uptake
due to siderophore production by rhizobacterial inoculants (Rajkumar et al., 2010).
The role of siderophores produced by fluorescent pseudomonads in plant growth
promotion was first reported by Kloepper et al. (1980a). Fluorescent Pseudomonas
produces siderophores, referred to as pyoverdines. These are yellow-green pigments that
fluoresce under UV light and are able to chelate iron present in the soil, thereby
depriving pathogens of iron necessary for their growth and pathogenesis (Leong, 1986).
Pyoverdins are known to be involved in the suppression of phytopathogens (Bakker et
al., 1986 and Becker and Cook, 1988). Competitive exclusion of pathogen by forming
iron-siderophore complexes plays a vital role in biocontrol of several phytopathogens
such as Fusarium oxysporum (causal agent of wilt), Pythium ultimum (causal agent of
damping off of cotton) and Pythium sp. (causal agent of root rot of wheat) (Kloepper et
al., 1980b; Scher and Bakker, 1982 and Becker and Cook, 1988). Rhizobacteria produce
various types of siderophores (Pseudobactin and ferrooxamine B) that chelate the
scarcely available iron and thereby prevent pathogens from acquiring iron (Loper and
Buyer, 1991).
Inhibition of phytopathogens such as F. oxysporum f. sp. cubense, F. oxysporum
f. sp. vasinfectum, Rhizoctonia solani and Acrocylindrium oryzae by various strains of P.
fluorescens that exhibited siderophore production has been reported (Sakthivel et al.,
1986). It can be stated that fluorescent pseudomonads by their ability to form ferric-
siderophore complex prevents the availability of iron rendering it inaccessible to other
50
microorganisms (Leong, 1986). Plant growth promoting fluorescent Pseudomonas sp.
RBT 13 exhibited significant siderophore production and was found effective against
several fungal and bacterial phytopathogens (Dileep Kumar and Dubey, 1993).
Siderophore production was observed for disease suppression by fluorescent
Pseudomonas WCS358 on radish, carnation, and flax using various Fusarium oxysporum
strains as the pathogen. The study presented that siderophore production exhibited by the
wild-type strain was found to be more effective than the mutant defective strain
(Raaijmakers et al., 1995).
Enhanced plant growth of lentil by five strains of fluorescent pseudomonads was
reported through siderophore production as a mechanism for biocontrol of wilt caused by
F. oxsporum f. sp. lini (Rao et al., 1999). It has been reported that Pseudomonas
aeruginosa strain IE-6 and its streptomycin resistant strain IE-6S+ significantly
suppressed nematode Meloidogyne jauanica population densities in tomato root thereby
reducing subsequent root knot development (Siddiqui and Shaukat, 2003). Strong
antifungal activity against various fungal phytopathogens such as Aspergillus niger, A.
flavus, A. oryzae, F. oxysporum and Sclerotium rolfsii were reported by purified
siderophores obtained from Pseudomonas strains (Manwar et al., 2004). Mostly
siderophores are part of primary metabolism for growth and development, in few cases
these may considered as antibiotics (secondary metabolites) (Haas and Defago, 2005).
Siderophore production is influenced by various environmental factors such as
pH, the level of iron and the form of iron ions, the presence of other trace elements and
an adequate supply of carbon, nitrogen, and phosphorus (Duffy and Defago, 1999).
Different bacterial siderophores differ in their abilities to sequester iron (Loper and
Henkels, 1999). Few examples of siderophores and their producing organisms are listed
in table 3.
51
Table 3. Various kinds of Siderophore and their producing microorganisms
Siderophore Producing Microorganism Type of siderophore
Ferrichrome Ustilago sphaerogena Hydroxamate
Desferrioxamine B
(Deferoxamine)
Streptomyces pilosus
Streptomyces coelicolor
Hydroxamate
Desferrioxamine E Streptomyces coelicolor Hydroxamate
Fusarinine C Fusarium roseum and Hydroxamate
Ornibactin Burkholderia cepacia Hydroxamate
Enterobactin Escherichia coli Catecholate
Bacillibactin Bacillus subtilis
Bacillus anthracis
Catecholate
Vibriobactin Vibrio cholera Catecholate
Azotobactin Azotobacter vinelandii Siderophores with mixed
ligands
Pyoverdine Pseudomonas aeruginosa Siderophores with mixed
ligands
Yersiniabactin Yersinia pestis Siderophores with mixed
ligands
Jurkevitch et al. (1992) studied the differential availabilities of the hydroxamate
siderophores ferrioxamine B (FOB) and ferrichrome (FC) and the pseudobactin
siderophores as sources of Fe for soil and rhizosphere bacteria and found that the ability
of bacteria to utilize a large variety of siderophores confers an ecological advantage.
Catechol type of siderophore was produced by Acinetobacter calcoaceticus obtained
from wheat rhizosphere in black cotton soils of North Maharashtra (Prashant et al.,
2009).
Pseudomonas sp. are capable of utilizing heterologous siderophores that are
siderophores produced by diverse species of bacteria and fungi. One example is
Pseudomonas putida that utilizes siderophore produced by rhizosphere microorganisms
for enhanced iron availability in the natural habitat (Loper and Henkels, 1999). Some
PGPB strains were reported to derive iron from heterologous siderophores produced by
cohabiting microorganisms (Castignetti and Smarelli, 1986; Wang et al., 1993;
Raaijmakers et al., 1995; Loper and Henkels, 1999; Whipps, 2001 and Lodewyckx et al.,
2002). Siderophore producing Azotobacter vinelandii MAC 259 and Bacillus cereus UW
85 can be considered as efficient PGPR for enhancing crop yield (Husen, 2003).
Maximum yield of hydroxamate type of siderophore was obtained by Pseudomonas
fluorescens NCIM 5096 along with P. putida NCIM 2847 in the modified succinic acid
52
medium (SM) (Sayyed et al., 2005). (NH4)2SO4 and amino acids commenced bacterial
growth as well as siderophore production. Also, optimum siderophore yield was obtained
with urea.
Siderophore producing Bacillus megaterium isolated from tea rhizosphere
exhibited plant growth promotion and reduced disease intensity (Chakraborty et al.,
2006). Xie et al. (2006) performed analysis of siderophores (catechol) by polyamide
TLC. These methods have shown to be quite efficient for the sidero-analysis of P. putida
revealing the pyoverdine type of siderophore (Sarode et al., 2007). Hydroxamate-type of
siderophores were reported to be produced by Rhizobium strains isolated from the root
nodules of the Sesbania sesban (L) Merr. (Sridevi and Mallaiah, 2008). Studies suggest
that Rhizobium sp. and Mesorhizobium sp. are able to produce catecholate type of
siderophores (Joshi et al., 2009). Maximum siderophore production was reported fom E.
coli isolated from endorhizosphere of sugarcane (Saccharum sp.) and rye grass (Lolium
perenne) and promoted growth of plants (Gangwar and Kaur, 2009). Siderophore
producing abilities of Bacillus and Pseudomonas were evaluated in the presence and in
absence of technical grade of herbicides such as quizalafop-p-ethyl and clodinafop
(Munees and Mohammad, 2009). It was found that the metabolic activities of both the
rhizobacteria were high in the absence of herbicides and declined in the presence of
herbicides.
2.3.5.6 ACC Deaminase Activity
Ethylene is an essential metabolite for plant’s growth and development. It plays
a vital role in inducing physiological changes in plants (Khalid et al., 2006). Nearly all
plants produce ethylene endogenously influenced by various biotic and abiotic processes
in soils. These endogenous levels of ethylene are known to increase during various
stress conditions caused by salinity, drought, water logging, heavy metals and
pathogenicity. Also, referred as stress hormone, such increased levels of ethylene impart
negative effect on the overall plant health (Saleem et al., 2007). Elevated levels of
ethylene may adversely affect plant health such as defoliation in crops (Saleem et al.,
2007 and Bhattacharyya and Jha, 2012).
Few PGPR may induce salt tolerance and reduce drought stress in plants. These
PGPR possess the enzyme ACC deaminase capable of decreasing ethylene levels,
53
thereby facilitating plant growth and development (Nadeem et al., 2007 and Zahir et al.,
2008). A wide range of bacterial genera exhibits ACC deaminase activity. Examples
include Acinetobacter, Achromobacter, Alcaligenes, Azospirillum, Bacillus,
Enterobacter, Pseudomonas, Rhizobium, Serratia etc. (Shaharoona et al., 2007a, b;
Nadeem et al., 2007; Zahir et al., 2008 and Kang et al., 2010).
ACC deaminase possessing rhizobacteria take up ACC, which is the ethylene
precursor and converts it into 2-oxobutanoate and ammonia (Arshad et al., 2007).
Different stress conditions that are relieved by ACC deaminase containing PGPR
includes phytopathogenecity stress from drought, polyaromatic hydrocarbons, radiation,
heavy metals, high levels of salt, extreme temperature conditions, flooding, etc.
(Lugtenberg and Kamilova, 2009 and Glick, 2012). Such rhizobacteria promotes various
plant growth parameters, mycorrhizal colonization, nutrient uptake and nodulation
efficiency among different crops (Nadeem et al., 2007; Shaharoona et al., 2008 and
Glick, 2012).
According to the model outlined by Glick et al. (1998), major portion of ACC
exuded from plant roots/seeds is taken up by the soil microbes or hydrolyzed by
microbial enzyme ACC deaminase yielding ammonia and α-ketobutyrate, leading to
decreased ACC levels outside the plant. Exudation of ACC in the rhizosphere balances
the internal and the external ACC levels. Due the presence of microbial ACC deaminase
activity, plants tend to synthesize greater amounts of ACC than required by the plant.
This causes release of ACC from plant roots, exhibiting increased root growth. As a
result, microbial population around the plant roots increases due to availability of ACC
as nitrogen source. Further, lower ACC levels in plant inhibit ethylene biosynthesis. A
schematic representation of this model is shown in Fig. 2.
54
Fig 2 Model of action of bacterial ACC deaminase
(Courtesy by: Tarun et al., 1998)
Certain PGPR contain a vital enzyme, ACC deaminase, which regulates ethylene
production by metabolizing ACC (an immediate precursor of ethylene biosynthesis in
higher plants) into α-ketobutyrate and ammonia. PGPR possess several other traits like
synthesis of auxins, gibberellins, cytokines and/or polyamines, which directly promote
plant growth (Tabor and Tabor, 1985; Frankenberger and Arshad, 1995; Patten and
Glick, 2002 and Zahir et al., 2004). Inoculation with PGPR containing ACC deaminase
activity could be helpful in sustaining plant growth and development under stress
conditions by reducing stress-induced ethylene production (Saleem et al., 2007).
(i) ACC deaminase PGPR and stress
Bensalim et al. (1998) revealed that a plant growth promoting rhizobacterium
Burkholderia phytofirmans strain PsJN helped potato plants in maintaining normal
growth under heat stress. Grichko and Glick (2001) studied the effect of inoculation
with ACC deaminase PGPR Enterobacter cloacae CAL2, Pseudomonas putida UW4,
P. putida (ATCC17399/pRKACC) or P. putida (ATCC17399/pRK415) on tomato
subjected to flooding. According to the study, it has been reproted that plants inoculated
with PGPR containing ACC deaminase were better able to thrive through the salinity
stress while demonstrating a normal growth pattern. In this direction, Mayak et al.
(2004b) reported increased fresh and dry weights of both tomato and pepper seedlings
exposed to transient water stress by ACC deaminase PGPR A. piechaudii ARV8. In a
55
study conducted by Mayak et al. (2004a), it was observed that Achromobacter
piechaudii possessing ACC deaminase activity enhanced the fresh and dry weights of
tomato seedlings under salt stress. Similar results have been observed in the case of
maize growth under salt stress in response to inoculation with ACC deaminase PGPR
(Nadeem et al., 2006).
Recently, Cheng et al. (2007) has also reported a psychrotolerant ACC
deaminase bacterium P. putida UW4 can promote canola plant growth at low
temperature under salt stress. Saravanakumar and Samiyappan (2007) reported
Pseudomonas fluorescens strain TDK1 containing ACC deaminase activity can enhance
the saline resistance in groundnut plants and increased yield as compared with that
inoculated with Pseudomonas strains lacking ACC deaminase activity. Upadhyay et al.
(2011) studied plant growth-promoting attributes of the salt tolerant isolates from wheat
rhizosphere, exhibiting EPS producing potential at different NaCl concentrations (0, 30
and 60 g/l) and concluded that salt-tolerant plant growth-promoting rhizobacteria
(PGPR) can play an important role in alleviating soil salinity stress during plant growth
and bacterial exopolysaccharide (EPS) can also help to mitigate salinity stress by
reducing the content of Na+ available for plant uptake. Also, isolates showed plant
growth promoting traits such as IAA production, siderophore activity and ACC
deaminase activity.
(ii) ACC deaminase PGPR and Biocontrol
Yuquan et al. (1999) reported isolation of ACC deaminase bacteria that showed very
strong antagonism against plant pathogen Fusarium oxysporum. Donate-Correa et al.
(2005) have also reported the positive effect of ACC deaminase bacterium P.
fluorescens on C. proliferus (tagasaste) in antagonizing the growth of Fusarium
oxysporum and Fusarium proliferatum in growth medium. Pandey et al. (2005) noted an
ACC deaminase containing Burkholderia sp. for strong antagonistic activity against R.
solani and S. sclerotiorum. Contrarily, Rasche et al. (2006a) found no antagonistic effect
of ACC deaminase bacteria against bacterial pathogen Erwinia carotovora sp.
atrospetica (Eca). Interestingly, in another study, Rasche et al. (2006b) reported that
ACC deaminase bacteria were also capable of antagonizing at least one of the two
potato pathogens Ralstonia solanacearum and Rhizoctonia solani. Belimov et al. (2007)
concluded that bacterial ACC deaminase of Pseudomonas brassicacearum Am3
56
(pathogenic bacteria) can promote growth in tomato by masking the phytopathogenic
properties of this bacterium only at lower concentration.
(iii) ACC deaminase PGPR and phytoremediation
Reed and Glick (2005) studied the growth of canola (Brassica napus) seeds treated with
PGPR in copper-contaminated and creosote-contaminated soil. Arshad et al. (2007)
have recently investigated the significance of PGPR containing ACC deaminase activity
in improving the growth of plants in the presence of organic contaminants and its
application in phytoremediation of heavy metal contaminated soil environment. Cavalca
et al. (2010) isolated Arsenic-resistant bacteria associated with roots of the wild Cirsium
arvense plant from an arsenic polluted soil and screened for potential plant growth-
promoting characteristics. It was found that several isolates were able to reduce arsenate
and to oxidise arsenite. Ancylobacterdichloromethanicum strain As3-1b possessed both
characteristics. Some rhizobacteria produced siderophores, indole acetic acid and ACC
deaminase, thus possessing potential plant growth-promoting traits. So-Yeon et al.
(2010) studied plant growth-promoting traits includingACC deaminase activity of
rhizobacteria isolated from rhizoplane or rhizosphere of C. leiorhyncha, E. crusgalli, C.
communis, E. arvense, C. kobomugi, P. lapathifolia occurring in contaminated soil with
petroleum and heavy metals and found that the ratio of PGPT-possessing rhizobacteria
in the rhizoplane was higher than that in the rhizosphere.
(iv) ACC deaminase PGPR and Increased Nodulation
Cattelan et al. (1999) reported that ACC deaminase rhizobacteria caused early growth
and promoted nodulation in soybean. Recent studies have also demonstrated that genetic
modification of PGPR expressing ACC deaminase genes helped in modulation of
nodulation in legumes and biological control of plant disease (Wang et al., 2000; Ma et
al., 2003). Ma et al. (2003) found that ACC deaminase rhizobacterium Rhizobium
leguminosarum bv. viciae 128C53K enhanced the nodulation in Pisum sativum cv.
sparkle by modulating ethylene levels in the plant roots during the early stages of nodule
development. Okazaki et al. (2004) has reported that PGPR containing ACC deaminase
for increasing nodulation efficiency in legumes. Shaharoona et al. (2006) reported that
co-inoculation with Bradyrhizobium plus ACC deaminase rhizobacteria increased
nodulation in mung bean compared to inoculation with Bradyrhizobium only.
57
2.3.5.7 Production of volatile organic compounds
This is one of the important mechanisms to stimulate the growth of plants. Also, it is a
strain-specific phenomenon. Farmer (2001) identified low-molecular weight plant
volatiles, terpenes and jasmonates as potent signal molecules. Crops such as tobacco,
carrot, maize and rice were reported for acetoin-forming enzyme (Forlani et al., 1999).
These volatiles may act as signaling molecules to mediate plant–microbe interactions as
(Ryu et al., 2003). Production of volatiles has been reported by several PGPR strains (B.
subtilis GB03, B. amyloliquefaciens IN937a and E. cloacae JM22) (Ryu et al., 2003).
These strains released volatile components, such as, 2, 3-butanediol and acetoin. This
resulted in growth of Arabidopsis thaliana. In the absence of physical contact with plant
roots, volatile chemicals released from specific strains of PGPR can also trigger growth
promotion and induced resistance in the model plant Arabidopsis (Ryu et al., 2003).
Bacillus subtilis GB03 emits a complex blend of volatile components that stimulates
promotion of plant growth. It is a commercially available saprophytic symbiont. It is
reported that Bacillus subtilis GB03 releases volatile chemicals that elevated fresh
weight essential oil accumulation and emissions along with plant size in the terpene-rich
herb sweet basil (Ocimum basilicum). In plants exposed to GB03 volatiles or with root
inoculation, the weight (fresh and dry) and essential oil components enhanced
considerably. The study suggests the role of bacterial volatiles for increased oil
production and as well as biomass in sweet basil (Banchio et al., 2009).
2.3.5.8 Heavy Metal Tolerance
Metals and metalloids that have densities greater than 5 g cm-3
are considered as heavy
metals. Some of these (essential metals) are necessary at low concentrations by
organisms. These metals are also found to be associated with pollution and toxicity
(Adriano, 2001). Zinc (Zn) plays an important role in the metabolism of carbohydrates,
proteins, phosphate, auxins; formation of RNA and ribosome in plants and acts as a
component of many enzymes such as dehydrogenases, proteinases, peptidases, etc.
(Mengel and Kirkby, 1982 and Kabata-Pendias and Pendias, 2001). Copper (Cu) is
necessary for several physiological processes in plants such as photosynthesis,
respiration, nitrogen and cell wall metabolism. It also contributes in providing disease
resistance (Kabata-Pendias and Pendias, 2001). Both these metals are of significance for
proper functioning of human as well as bacterial metabolism. Also, these metals may be
toxic to cells at high concentrations (Baker and Walker, 1989 and Cavet et al., 2003).
58
Metals that do not play role in known biological processes are referred as non
essential metals. For example, cadmium (Cd) may be quite toxic as it is accumulated by
organisms. The toxic effects due to cadmium includes disturbances in activity of
enzymes, inhibition of DNA-mediated transformation in microorganisms, increased plant
predisposition to fungal invasion and interference in symbiosis between microbes and
plants (Kabata-Pendias and Pendias, 2001). Cadmium may assists disorders of Ca and
vitamin D metabolism causing bone degeneration and kidney damage (itai-itai disease)
in humans (Adriano, 2001). The continuous accumulation of heavy metals in the food
chain results in their excessive uptake by animals and humans. During last few decades,
heavy metal pollution and its effects have gained much attention. Heavy metal pollution
is a significant problem worldwide, due to its effects on 12% of the world’s agricultural
land (Moffat, 1999).
Heavy metals such as Cu, Zn and Cd in soils cannot be biodegraded and
accumulates in the soil. Heavy metals may find their way into the soil from various
sources like volcanic eruptions, weathering of rocks, mines, pesticides, industrial
processes, fossil fuel combustion etc. (Alloway, 1995 and Ernst, 1998). Fertilizers are
also responsible for increasing heavy metal concentrations into the soil. It has been
reported that cadmium uptake of wheat enhanced due to addition of phosphatic fertilizers
(Hamon et al., 1998). The chemical speciation of heavy metals decides their
bioavailability and risks emerging due to these heavy metals (Adriano, 2001).
Due to the increased introduction of pollutants into groundwater and soil, the
self-cleaning capacity of the ecosystem gets disturbed and leads to their accumulation.
Due to the exceeding harmful levels of heavy metals, the soil is becoming unsuitable for
sustainable agriculture. The increasing exposure to toxic heavy metal is a potential threat
for both human and plant health (Aziz et al., 1996). Metal toxicity may lead to different
negative effects such as inactivation of plant proteins (Assche and Clijsters, 1990),
decline in rhizobial populations (Chaudri et al., 2000) and decreased nitrogen fixation
(Broos et al., 2005).
Numerous mechanisms have been devised by the rhizosphere microorganisms for
prevention of stress commenced due to heavy metal contamination. These include
59
formation and sequestration of heavy metals, reduction of toxic metal to less toxic forms
and direct efflux of metal out of the cell (Outten et al., 2000). Among the rhizosphere
microorganisms, PGPR are known to increase plant growth and yield and reduce heavy
metal toxicity (Gupta et al., 2004; Tripathi et al., 2005 and Wu et al., 2005).
Elevated levels of heavy metal reduce microbial activity in soil and soil fertility.
Accumulation of nickel in soil reduces agricultural yield. From soil, it enters the food
chain and adversely effects health (Guo et al., 1996 and Salt et al., 1999). It is strongly
phytotoxic at elevated concentrations (Boominathan and Doran, 2002). Nickel is used in
various industries all over the world. During various industrial operations, a considerable
amount of nickel is released into atmosphere and soil. Nickel toxicity occurs due to
binding of nickel to sulf-hydryl groups of sensitive enzymes or displacing essential metal
ions in various biological processes (Valko et al., 2005 and Cheng et al., 2009).
Cadmium is a highly toxic pollutant that is excessively hazardous to the
environment. It causes damage to biodiversity and leads to reduction in soil microbial
activity (McGrath, 1994 and Chen et al., 2003). It causes inhibition of root and shoot
growth and reduction in nutrient uptake (Sanita di Toppi and Gabrielli, 1999). Studies
suggest that prolonged exposure of heavy metals leads to reduction in soil microbial
diversity and decreased metabolic processes (Smith et al., 1997 and Kojdroj and Van
Elsas, 2001). Due to its accumulation of Cd in soil, it passes through the plant into the
food chain and causes diseases in humans. Cadmium finds its way into water bodies
from various sources such as smelting, cadmium-nickel batteries, phosphatic fertilizer,
sewage sludge, stabilizers, alloy industries and mining (Nanganuru and Korrapati, 2012).
About 59% of P. syringae pv. Syringae isolates obtained exhibited copper
resistance and wer found resistant to cupric sulfate (Cazorla et al., 2002). Pseudomonas
sp. C-171 showed tolerance to hexavalent chromium (Cr+6
) up to 2000 ppm as potassium
dichromate (Rahman et al., 2007). About 90% of chromium was reported to be degraded
by Pseudomonas SP8 (Poornima et al., 2010). A metal resistant, PGPR bacteria
Pseudomonas aeruginosa PDMZnCd2003 exhibited different mechanism response in Cd
as compared to Zn and Zn+Cd (Meesungnoen et al., 2012).
60
Several studies have been conducted to study the negative effects imparted by
heavy metals on microbe-mediated processes (Duxbury, 1985; Baath, 1989 and Giller et
al., 1998). The negative effects include reduced mineralization of carbon and fixation,
decrease in nitrogen transforming ability, reduced soil enzyme activities, decreased
microbial numbers (cfu) and increased frequency of heavy metal resistant bacteria
(Doelman et al., 1994; Pennanen et al., 1996 and Muller et al., 2001).
PGPR strain Kluyvera ascorbata SUD165 inoculated to Indian mustard and
canola seeds, exhibited siderophore production and ACC deaminase activity and
rendered resistance against Ni, Pb and Zn toxicity to the plants (Burd et al., 1998).
Immobilization of cadmium leading to increased growth and nutrient uptake in barley
plants by nitrogen-fixing and auxin-producing PGPR in the presence of toxic Cd
concentrations were reported (Belimov and Dietz, 2000). It was reported that metal-
resistant PGPR containing ACC deaminase commenced plant growth on inoculation in
rape (canola) in soils contaminated with cadmium (Belimov et al., 2001).
2.3.5.9 Microbial Antagonism
Biocontrol agents can be described as bacteria that aids in reducing plant disease
incidence/severity. On the other hand, antagonists are those bacteria that exhibit
antagonistic activity towards a pathogen (Beattie, 2006). Bacterial antagonistic activities
includes synthesis of hydrolytic enzymes (chitinases, glucanases, proteases and lipases)
that lyse pathogenic fungal cells, competition for nutrients and colonization of niches at
the root surface, regulation of plant ethylene levels through the ACC-deaminase enzyme
for stress resistance, production of siderophores and antibiotics (Glick and Bashan, 1997
and Van Loon, 2007).
Rhizobacteria are suitable candidates for use as biocontrol agents. These
rhizobacteria inhabit the rhizosphere and through their antagonistic activity protects the
plant before and during primary infection of roots caused by diverse plant pathogens.
PGPRare native tosoiland plantrhizosphere and possess the ability to control or inhibit a
broadspectrum ofbacterial, fungal andnematodediseases and because of their contribution
towards plant growth and protection against pathogens, the use of PGPR has increased
all over the world. This application of PGPR holds a great significance for agriculture for
biocontrol of plant pathogens and biofertilization (Siddiqui, 2006). Multiple PGP traits
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were found to be exhibited by bacterial strains isolated from Lolium perenne rhizosphere
that may act as suitable PGPR and biocontrol agents (Shoebitz et al., 2007). Suppression
of phytopathogens by PGPR leading to enhanced plant growth may occur through
different mechanisms such as production of antibiotics, fungal wall-lysing enzymes or
hydrogen cyanide. Pseudomonas mediated antagonistic microbe-microbe interactions
aids in the biocontrol of phytopathogenic fungi in the rhizosphere contributing in
enhanced plant growth and survival (Winding et al., 2004). According to Costa et al.
(2007), antagonistic interactions involves synthesis of various antibiotics like 2, 4-
DAPG, pyoluteocin etc.
2.3.6 Characterization of PGPR strains
A PGP isolate Pantoea NII-186 was isolated from soil sample of Western Ghat Forest
and characterized (Dastager et al., 2009). The strain showed strong phosphate
solubilizing activity. The isolate was found to be Gram-negative and rod shaped. It
produced round, light brown and translucent colonies on nutrient agar plates. The isolate
showed positive test for nitrate reduction, tween 80 and 40 degradation, negative for
H2S, utilization of casein and starch, hydrolysis of gelatin, phenol degradation, and
cellulose. The starin was identified by 16S rRNA gene sequencing and showed 99%
similarity to the sequence of Pantoea agglomerans LMG 1286. Another potent
phosphate solubilizing bacterial strain NII-0909 isolated from the Western ghat forest
soil was characterized (Dastager et al., 2010). It was found to be Gram-positive, non –
motile and coccus shaped. Colonies formed by the strain were circular in shape having
smooth suface with entire margin and pale yellow in colour. It gave positive test for
reduction of nitrate degradation of tween 80. The isolate displayed negative test for H2S,
casein utilization, gelatin hydrolysis, phenol degradation and utilized carbon sources
such as sorbitol, L-Arabinose, melibiose, inositol, mannitol and rhamnose. It was
identified by 16S rRNA gene sequencing and was found to belong to Micrococcus
genus.
About 11 rhizobacterial isolates obtained from lentil rhizosphere were subjected
to their morphological and biochemical characterization (Saini, 2012). These strains were
screened for their antifungal activity against plant pathogen Fusarium oxysporum. About
36.3% isolates exhibited yellow to green pigmentation on King’s B medium. These
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isolates were found to be Gram-negative and showed positive test for indole, citrate,
catalase and negative for methyl red. These isolates were found to belong to genera
Pseudomonas. Among these eleven isolates, seven isolates were recorded as Gram
positive rods and showed positive test for indole, catalase, citrate utization and nitrate
reduction. These seven isolates were found to belong to genera Bacillus. Yildiz et al.
(2012) screened Bacillius and Pseudomonas rhizobacteria against Fusarium oxysporum
f. sp. melongenae that causes wilt disease in eggplant. These isolates were tested for their
PGP traits. The isolates were morphologically as well as biochemically characterized.
Colony colours varied from cream to white. Few isolates showed coloured colonies such
as yellow, orange, pink, green and light brown. They showed negative catalase test and
hypersensitive reaction on tobacco leaves.
About 96 rhizobacterial isolates obtained from A. angustifolia were subjected to
tests for the identification of their multiple PGP attributes (Ribeiro and Cardoso, 2012).
Among them, 28 isolates were obtained from King’s B medium and 68 from potato
dextrose agar medium. Abouty 21 isolates that showed the maximum number of growth
promotion traits were subjected to FAME analysis. FAME analysis revealed presence of
bacteria belonging to Bacillaceae, Enterobacteriaceae and Pseudomonadaceae families.
Among these, one isolate belong to genera Pseudomonas, three isolates belong to
Ewingella and eight isolates belong to Bacillus. About ten bacterial isolates were
obtained from the rhizosphere soils of rice field from Kashipur and were characterized
(Sharma et al., 2012). They were designated as PGB1 to PGB10. The morphological
characteristics of these isolates widely varied. All the isolates produced round shaped
and raised colonies having smooth shiny surface with smooth margin. All isolates were
odourless and no pigmentation in colonies was observed. Size of the colonies varied
from 0.2 to 2 mm. Among them, seven isolates were found to be rod shaped. PGB2 and
PGB5 were ellipsoidal in shape, PGB8 was coccus in shaped. All the isolates exhibited
motility and showed Gram negative reaction. About 60 rhizobacterial isolates were
isolated from the rhizosphere of Withania somnifera (Rathaur et al., 2012b). Isolates
PG1, PG3, PG5, PG7, PG9 and PG10 were selected for further study. All the isolates
were gram positive. rod shaped and formed smooth, raised, shiny colonies. Colony size
varied from 0.2-2.0 mm in diameter. They showed positive test for oxidase, catalase and
starch hydrolysis.
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About 28 bacterial strains were isolated from rhizospheric soil sample of various
locations of fourteen rice fields of districts of Nepal and were characterized (Shrivastava,
2013). All isolates showed gram negative test. All the isolates were found to be positive
for catalase and nitrate reduction test. About 19 isolates showed positive test for urease
and motility, 10 were positive for methyl red test, 18 isolates were positive for citrate
utilization test and 8 were positive for lactose fermentation test. Biochemical
characterization revealed that the isolates belonged to genera Agrobacterium,
Azotobacter, Klebsiella, Pseudomonas and Serrattia. About 16 endophytic bacteria
obtained from polyembryonic mango root stock (GPL-3 and ML-4) were characterized
both morphologically and biochemically. Colony of the isolates varied from yellowish to
creamy yellow and white to creamy white in color (Kannan et al., 2014). Few isolates
produced yellow to greenish yellow pigmentation. About 60.75% of the isolates were
recorded as Gram-positive and 31.25% were recorded as Gram negative bacteria. Most
of the isolates were found to be rod shaped. About 140 bacterial isolates were obtained
from plant rhizosphere soils from Khammam districts (Geetha et al., 2014a). These
isolates were evaluated for their antagonistic potential and PGP traits. Six best bacterial
strains were chosen for characterization. The PGPR isolates viz., KG-50, BG-72, TG-60,
MG-58, MG-64 and WG-57, showed wide variation in their morphological
characteristics. Colonies were found to be round, raised with smooth margins. Few
colonies were rough and undulated whereas as others were shiny smooth surfaces. None
of the colonies showed pigmentation. All isolates were rod shaped, motile, two were
found to be Gram positive and four were Gram negative.
2.3.7 Optimization of cultural conditions
A psychrotolerant PGPB was isolated from North Western Indian Himalayas.
The bacterium was identified by morphological, biochemical and 16S rRNA gene
sequencing as Pseudomonas sp. The strain was found to be Gram negative and rod
shaped. Studies were conducted to check the effect of different temperature, pH and salt
concentrations on the growth ability of this strain. This strain was able to grow over a
wide temperature range of 4 to 35 °C, but maximum growth was found to occur at 28 °C.
It had a pH range of 5 to 10 (optimum pH 7.0) and could tolerate NaCl up to 5% (w/v).
Positive carbon sources were D-arabinose, L-arabinose, galactose, glucose, malonate,
mannose, melibiose and xylose, utilization (Mishra et al., 2008). Ten bacterial isolates,
namely, PGB1 to PGB5, PGT1 to PGT3, PGG1 and PGG2, were isolated from
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rhizosphere soils various locations of Mymensingh in Bangladesh. and characterized. It
was also noted that the growth of isolates on LB agar plates varied in temperature. The
growth of all isolates was good in the temperature ranges of 20 to 28 °C. In addition,
PGB3 and PGB4 isolates were found to grow at 45 °C (Ashrafuzzaman et al., 2009).
PGPR strain Pantoea NII-186 was isolated from Western Ghat Forest soil, India and
characterized. The ability of the isolate to grow in diverse temperature range i.e. 5 °C to
40 °C (at a 5 °C interval); pH range 4.0 to 12.0 (at a pH 1.0 interval) and 0-25% (w/v)
NaCl concentrtions was evaluated. It was able to grow over a wide range of temperature
5 °C to 40 °C, with optimum at 28 °C to 30 °C. It had a pH tolerance over the range of 4-
11, with optimum 7.0 ± 0.5 and could tolerate 7% of NaCl concentration (w/v) (Dastager
et al., 2009).
Pseudomonas fluorescens strain CV6 isolated from cucumber rhizosphere in
Varamin was tested for its ability to grow on carbon sources such as L-arabinose, D-
galactose, trehalose, meso-inositol and sorbitol. The strain CV6 exhibited growth on all
these carbon sources (Maleki et al., 2010). Datta et al. (2010) isolated and characterized
bacterial isolates (Pseudomonas and Xanthomonas) from chilli rhizosphere and tested
their PGPR potential. They also tested growth on carbon sources such as arabinose,
cellobiose, dextrose, fructose, lactose, inositol, lactose, mannitol, moltose, raffinose and
sucrose was tested. Most of the isolates preferred to use simple sugar, such as dextrose or
fructose. Few isolates such as C3, C6, C22, C23, and C36 however, preferred sugar
alcohol such as mannitol or inositol also with various other sugars. The growth of the
isolates in different pH medium (4, 6, 8, 10 and 12) was recorded. In contrast, all the
isolates had a wide range of pH tolerance. Although the very few test bacterial isolates
(only 12%) were able to grow at low pH; but all the other test isolates could grow well at
a pH range of 6 to 10 and about 56% of them could even withstand a pH of 12.
About 10 Azospirillum strains were isolated from paddy field rhizosphere soil of
Thanjavur district. They characterized the strains based on morphological characteristics
and biochemical tests such as IMViC, catalase, citrate, starch hydrolysis and urease test.
The effect of different temperatures (27 ºC, 32 ºC, 40 ºC, 45 ºC and 50 ºC) on these
isolates was noted and it was observed that all the strains were able to grow at different
temperatures successfully. The ability of these strains to grow in different pH range was
tested. Four isolates, namely, Azo2, Azo3, Azo4 and Azo5 exhibited growth in different
65
pH range, i.e. 5.5, 6.0, 6.8, 7.0 and 7.5. Other isolates, viz., Azo1, Azo6, Azo7, Azo8,
Azo9, Azo10 did not showed any growth at pH 5.5. Also, at pH 6.0, Azo6 and Azo1
strains were not able to grow. All the 10 strains utilized mannitol, fructose and succinate.
But only one strain could not utilize sucrose (Azo4) and lactose (Azo6) (Usha and
Kanimozhi, 2011). The indigenous isolates of PGPR were isolated from cauliflower
rhizosphere soil in different agro-climatic zones of Himachal Pradesh (Kaushal et al.,
2011). Five efficient isolates designated as MK2, MK4, MK5, MK7 and MK9 were
selected and characterized after successful experiments under in vitro and net house
conditions at varying levels of N and P on the growth and yield of cauliflower. Isolate
MK5 was found to be the most promising with maximum PGP traits. The pH range for
the isolate was 5 to 8. In our study, the optimum pH range for our selected isolates was
found to be 5 -7.5.
Rathaur et al. (2012b) studied the effect of UV-B tolerant PGPR on seed
germination and growth of Withania somnifera. The isolates were checked for their
growth under different temperatures (10 °C, 20 °C, 28 °C, 37 °C, 45 °C). The growth of
all isolates was good in the temperature range of 20 °C to 28 °C. In addition, PG3
showed maximum tolerance to temperature (45 °C). PGPR isolates obtained from rice
plants rhizosphere in Kashipur region were subjected to test for growth under different
temperatures such as 10 °C, 20 °C, 28 °C, 37 °C and 45 °C. Most of the isolates showed
growth within temperature range of 20 °C to 37 °C. Eight isolates showed growth at 10
°C (except PGB 8, PGB 10). Only two isolates i.e., PGB3 and PGB4 were able to grow
at 45 °C (Sharma et al., 2012). Four Bacillus strains, AM1, D16, D29 and H8, isolated
from tomato and potato rhizosphere have shown high potential of antagonistic activity
against the pathogen in laboratory and greenhouse experiments. These strains were
evaluated for growth in different pH values, growth in different NaCl concentrations and
utilization of some carbon sources including sucrose, L-rhamnose, arginine, glycerol,
lactic acid, inositol, D-rhafinos and D-Sorbitol. All the four isolates were able to grow in
different salt concentrations such as 1%, 4% and 8% NaCl. Strain D29, AM1and D16 did
not grow on pH 4 and 5. The optimum pH for growth was found to be 6. Only Bacillus
strain H8 was able to grow at pH 4, 5 and 6. The biochemical characterization, 16S
rRNA gene sequence fatty acid methyl esters analysis revealed the strains AM1 and D29
as Bacillus amyloliquefaciens, D16 as Bacillus subtilis and H8 as B. methylotrophicus,
respectively. For carbon utilization, D29 showed positive test for arginine, inositol, lactic
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acid, sucrose, D-sorbitol and D-rhafinos. AM1 (positive for arginine, inositol, glycerol,
lactic acid, sucrose, D-sorbitol and D-rhafinos); D16 (positive for arginine, lactic acid,
sucrose, D-sorbitol) and H8 (positive for glycerol, lactic acid, D-sorbitol and D-rhafinos)
showed varied carbon utilization abilities (Almoneafy et al., 2012).
Kaushal et al. (2013) studied the plant growth promoting traits of Pseudomonas
sp. in Oryza sativa. The isolated Pseudomonas sp. was tested for growth ability under
various pH (4, 5, 6, 7, and 8) and temperature conditions (28 °C, 30 °C, 32 °C, 34 °C and
36 °C). The isolate showed maximum growth at pH 6 at temperature 30 °C. Four PGPR
bacterial isolates (AK1, AK2, AK3 and AK4) were successfully isolated, characterized
from cauliflower roots and identified as belonging to genera Azospirillum, Pseudomonas
and Azotobacter. The isolates were tested for growth under different temperatures such
as 4 ºC, 12 ºC, 28 ºC and 37 ºC. The growth of all isolates was good in the temperature
range of 28 ºC to 40 ºC. In addition, AK3 and AK4 isolates showed maximum growth at
40 ºC (Kushwaha et al., 2013). About ten strains, three of them were nitrogen fixing (S1,
S2, S3, belongs to different genera of Pseudomonas, Aeromonas and Stenotrophomonas)
and six Azospirillum strains (Azo4, Azo5, Azo6, Azo7, Azo8, S9 and Azo10) isolated
from the rhizosphere of different regions in eastern Algeria were tested for growth at
different temperatures, pH range, salt tolerance and their ability for carbon source
utilization. It was noted that the strains isolated have a good growth in the five different
temperatures used (27 °C, 32 °C, 40 °C, 45 °C and 50 °C), but some strains (Azo8,
Azo10, S3 and S9) do not have growth at 50 °C. Thus, some isolates (S1, S2, Azo5,
Azo7, Azo8, S9 and Azo10) have a good growth and tolerate different pH levels (5.5,
6.0, 6.8, 7.0 and 7.5). However, isolates S3, Azo4 and Azo6 do not tolerate pH 5.5.
Concerning the use of different carbon source, majority of strains were able to use
mannitol, fructose, sucrose and lactose; but Azo5 does not use sucrose. Also, S3 and S9
does not use lactose.
The growth of the bacteria in the presence of different concentrations of NaCl
shows that the strains tolerate up to 300 mM NaCl. Also, strains S1, S2, Azo4, Azo5,
Azo7, Azo8 and S9 were very tolerant to the highest concentration of NaCl (700 mM)
but the growth rate was slightly lower compared to the other strains (S3 and Azo10).
They also recorded that the 10 selected isolates, S1, S2, Azo5, Azo7, Azo8 and Azo10
had a good growth at concentration of NaCl equal to 400 mM (Mahbouba et al., 2013).
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About 51 bacterial isolates were isolated from the rhizosphere soil samples of
different locations in Bangalore, Karnataka and screened for their antagonistic activity
against wide range of phytopathogens. Pseudomonas aeruginosa FP6 was found to be
the most promising candidate. Part of this study focused on the effect of NaCl,
temperature, and pH on growth of this strain. Bacterial growth study under stress
condition with respect to growth temperature, pH and salt (NaCl) was studied. The effect
of temperature range (20- 60 °C), pH range (5, 6, 7, 8, 9, 10) and NaCl concentration
(0.5M to 5M) was studied on the growth of bacterial isolate Pseudomonas aeruginosa
FP6. Strain Pseudomonas aeruginosa FP6 was able to grow on up to 4.5 M NaCl,
between 20 °C and 60 °C and at pH 5-10 (Bhakthavatchalu et al., 2013). Seven
Pseudomonas isolates (JUPF31, JUPF32, JUPF33, JUPF34, JUPF35, JUPF36, and
JUPF37) were isolated, characterized and tested for plant growth promoting traits, under
normal as well as saline conditions. These strains were obtained from soil samples of
different crops (rice, chilly, ragi, beans and garden soils) and were checked for their
ability to utilize different carbon sources such as sucrose, dextrose, mannitol and lactose.
It was noted that all the strains utilized these carbon sources to varying extent (Anitha
and Kumudini, 2014). Geetha et al. (2014b) isolated 180 PGPR strains from rhizosphere
soils of green gram and screened for their antifungal activity against Colletotrichum
capscici, Macrophomina phaseolina, Rhizoctonia solani and Fusarium oxysporum. Six
most effective isolates were tested for their temperature tolerance at 10 °C, 20 °C, 30 °C,
40 °C, 50 °C and 60 °C. All the selected Bacilli isolates were able to grow at 20 °C to 50
°C. At 60 °C and at 10 °C, the growth was not observed.
The optimum temperature was found to be 20 °C to 30 °C. About seven
phosphate-solubilizing bacterial strains were isolated and characterized for their
solubilization efficiency in Pikovskaya’s media amended with tri-calcium phosphate.
Only one bacterial strain recorded as a dominant phosphate solubilizer was identified as
Pseudomonas aeruginosa. The isolate was able to lower the pH and accumulate acid in
the medium. Acidification is the main mechanism of P solubilization. This strain was
further tested for its growth at different temperatures such as 10 °C to 50 °C for 4 days.
Although, growth was observed at different temperatures but the preferred temperature
was found to be 40 °C. The bacterial growth was also checked under different pH values
(2-10) and with different carbon sources. Among the tested pH, the maximum growth
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was registered in media adjusted in pH 7. The strain utilized different carbon sources
(fructose, lactose, glucose, galactose, maltose, sucrose, and arabinose. The most suitable
carbon source was found to be glucose. The highest growth was obtained in media
supplemented with casein within the nitrogen sources tested (Sankaralingam et al.,
2014). PGPR Stenotrophomonas maltophilia AVP27 isolated from the chilli rhizosphere
soil was subjected to test for growth different temperatures (25 ºC, 37 ºC, 50 ºC), pH
range (3, 5, 7, 9, 12), NaCl (0.3%, 0.5%, 0.7%, 0.9%, 1%), carbon sources (sucrose,
maltose, lactose, dextrose). The isolate AVP27 showed variation in growth at different
carbon sources. The maximum growth was recorded with glucose and minimum growth
with sucrose. Under different temperature range, moderate growth was observed at 25 ºC
and maximum at 37 ºC. Growth was recorded to be high at pH 7 and moderate at pH 9
(Kumar and Audipudi, 2015).
2.4 PLANT-MICROBIAL INTERACTIONS
Because the plant rhizosphere has high availability of nutrients, various soil
microorganisms prefer it to be their ecological niche. Growth promotion may be due to
other mechanisms such as production of phytohormones in the rhizosphere and other
PGP activities (Arshad and Frankenberger, 1993 and Glick, 1995). Depending on the
association of PGPR with their host plant, they can be of two types: intracellular PGPR
and extracellular PGPR. Intracellular PGPR are those located inside the specialized
nodular structures of root cells and extracellular PGPR, those exist in the rhizosphere, on
the rhizoplane or in the spaces between the cells of root cortex (Martinez-Viveros et al.,
2010). Intracellular PGPR includes endophytes such as Allorhizobium (de Lajudie et al.,
1998a), Azorhizobium caulinodans (Dreyfus et al., 1988), Bradyrhizobium japonicum
(Guerinot and Chelm, 1984), Mesorhisobium chacoense (Velazquez et al., 2001),
Mesorhizobium pluriforium (de Lajudie et al., 1998b), Rhizobium ciceri (Nour et al.,
1994), Rhizobium etli (Segovia et al., 1993), Rhizobium fredii (Scholla and Elkan, 1984),
Rhizobium galegae (Lindstrom, 1989) and Frankia species both of which can
symbiotically fix atmospheric nitrogen with the higher plants (Verma et al., 2010).
Examples of extracellular PGPR are Agrobacterium, Arthrobacter, Azotobacter,
Azospirillum, Bacillus, Flavobacterium, Micrococcous, Pseudomonas, Serratia.
There may be root–root, root-insect and root-microbe interactions, which leads to
greater production of plant exudates. The community structures of the rhizobacteria have
69
been studied through applications such as PCR and DGGE which showed alterations in
plant microbe interactions (Herschkovitz et al., 2005).
According to Whipps (2001), interactions between the rhizobacteria and growing
plants can be neutral, negative or positive. Most plant–associated rhizobacteria are
commensals and establish interaction with no visible effect on the host plant’s growth
and physiology (Beattie, 2006). Negative interaction is one where phytopathogenic
rhizobacteria produces negative impact on plant growth and physiology by production of
toxic substances such as hydrogen cyanide or ethylene whereas in positive interaction,
the rhizobacteria leads to plant growth enhancement through various mechanisms. Direct
mechanisms include nitrogen fixation, nutrient solubilization etc. whereas indirect
mechanisms include competitive exclusion of phytopathogens or by the removal of
phytotoxic substances (Bashan and de-Bashan, 2010).
Three different regions ae recognized, where plant–microbe interactions takes
place. These regions are: phyllosphere, endosphere and rhizosphere. Phyllosphere relates
to the aerial parts of the plants and endosphere with internal transport system.
Rhizosphere can be defined as that soil region which is largely influenced by the plant
roots and plant-produced material. It can also be defined as that region of soil bound by
plant roots, extending a few mm from the root surface and is much richer in bacteria than
surrounding bulk soil (Hiltner, 1904 and Bringhurst et al., 2001). Plant exudates in the
rhizosphere are major source of energy and nutrients and because of the rich availability
of nutrients, microbial populations are higher in this region (Haas and Defago, 2005).
Micro-organisms present in the rhizosphere play an important role in nutrient
cycling, (Cardoso and Freitas, 1992). Most important step towards use of micro-
organism as PGPR is is root colonization. Bacillus subtilis sp. and Pseudomonas sp. are
excellent rhizosphere colonizing bacteria (Steenhoudt and Vanderleyden, 2000 and
Trivedi et al., 2005). To be a successfull PGPR, strain must be capable of aggressive
colonization, plant growth stimulation and biocontrol (Weller et al., 2002 and Vessey,
2003). Rhizosphere colonization has been well documented in plants such as potato,
wheat, grasses, maize, and cucumber (Cakmakci et al., 2006). One mechanism for
rhizosphere colonization is siderophore production. Few examples include
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Bradyrhizobium japonicum, R. leguminosarum and S. meliloti (Carson et al., 2000 and
El-Tarabily and Sivasithamparam, 2006).
Whichever mechanism is used by PGPR for growth enhancement of plant, root
colonization is necessary (Glick, 1995). Actinomycetes, a major component of
rhizosphere microbiota are important source of different antimicrobial metabolites. They
help in nutrient cycling as well in plant growth-promotion (Halder et al., 1991; Elliot and
Lynch, 1995; Merzaeva and Shirokikh, 2006 and Terkina et al., 2006). Antagonistic
activity of endophytic Streptomyces griseorubiginosus against Fusarium oxysporum f.
sp. cubense has been recoreded (Cao et al., 2004). There have been reports of
rhizospheric Streptomycetes as biocontrol agent of Fusarium and Armillaria pine rot and
as PGPR of Pinus taeda (de Vasconcellos and Cardoso, 2009). Potential biocontrol
PGPR among Actinomycetes (Gomes et al., 2000 and Sousa et al., 2008) are
Micromonospora sp., Streptomyces spp., Streptosporangium sp., and Thermobifida sp.,
against root pathogenic fungi (Franco-Correa et al., 2010). In a study conducted on
endophytic actinomycetes present in Neem and Tulsi leaves for PGPR traits,
actinomycete isolate A7 (Streptomyces sp. mrinalini7) showed significant PGPR activity.
Isolate A7 was inoculated into model Tomato plant for assessment of its ability to
promote seed germination and plant growth and significant biomass production of
Tomato was recorded (Singh and Padmavathy, 2014).
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