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

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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

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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).