Chapter 2 Review of Literature

2.1. ABIOTIC STRESS

In the soil environment, a number of biotic and abiotic stresses affects the crop
productivity. Biotic stresses include plant pathogens and pests (viruses, bacteria,
fungi, insects, nematodes, etc.) and abiotic including salinity, drought, flooding,
heavy metals, temperature, gases and supplement lack or overabundance. As
compared to biotic stresses, abiotic stresses are the main source of yield reduction.
These stresses cause hormonal and nutritional imbalance in plants and physiological
disorders, for example, epinasty, abscission, senescence and susceptibility to diseases
(Nadeem et al., 2010). Abiotic stresses cause a particular direct or indirect negative
effect on plant growth and development (Figure 2.1). For instance, under salinity,
drought and water logging stress level of ethylene increases (Glick et al., 2007) that
inhibit the root growth and affect several plant processes (Belimov et al., 2002;
Sun et al., 2007). Drought stress not only enhances ethylene concentration but also
inhibit photosynthesis, causes change in chlorophyll contents and harms the
photosynthetic mechanical assembly. Because of restricted supply of water, root
development is seriously influenced. Similarly, other stresses like salinity, heavy
metals, nutrient deficiency or abundance and pathogen attack also cause negative
effect on plant growth and development. It disturbs hormonal balance, susceptibility
to diseases and causing metal toxicity (Glick et al., 2007; Saleem et al., 2007).

2.2. DROUGHT STRESS

Drought is one of the most significant abiotic stress factors, occurs when there is
severe loss of water in the soil and influences plant growth and productivity and leaf
photosynthesis (Dash and Mohanty, 2001; Flexas et al., 2004). A few plants have a
lot of physiological adjustment that permits them to endure water stress conditions.
Drought stress has become the most destructive abiotic stress over the past decades
and drastically affecting world’s food security. It ranges from moderate and short to
extremely severe and prolonged duration. Drought stress is a serious agricultural
problem which reduces crop productivity.

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Chapter 2 Review of Literature

Figure 2.1. Effects of different abiotic and biotic stresses on morphological, biochemical and physiological attributes of plants
(Kumar and Verma, 2018).

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Chapter 2 Review of Literature
Therefore, there is a dire need to find solutions to abate the consequences of drought
on agriculture productivity.

2.3. EFFECT OF DROUGHT STRESS ON PLANT GROWTH AND

DEVELOPMENT
Drought affects plant-water potential and interfere the normal functioning of plant by
changing physiological and morphological traits of plants (Rahdari and
Hoseini, 2012). Earlier in many studies it has been shown that drought stress causes
reduction in growth of several crops such as Hordeum vulgare L., Zea mays,
Oryza sativa and Triticum aestivum (Samarah, 2005; Kamara et al., 2003; Lafitte et
al., 2007; Rampino et al., 2006). Furthermore, it also influences the availability and
transport of nutrients to plant as water carries the nutrients from soil to the roots.
Therefore less water availability affects the nutrient diffusion and reduces flow of
water-soluble nutrients like calcium (Ca), magnesium (Mg), nitrate, sulphate, and
silicon (Si) (Selvakumar et al., 2012). Drought also activate antioxidant defense
system against ROS such as superoxide radicals (O 2•−), hydrogen peroxide (H2O2),
singlet oxygen (1O2) and hydroxyl radicals (OH•) that cause oxidative stress. In plants
accumulation of high ROS results in lipid peroxidation, membrane deterioration and
proteins, lipids and nucleic acids degradation in plants (Hendry, 2005;
Sgherri et al., 2000; Nair et al., 2008). Under drought stress, decrease in chlorophyll
content was observed in Paulownia imperialis, bean and Carthamus tinctorius
(Ayala-Astorga and Alcaraz-Meléndez, 2010; Siddiqi et al., 2009). Due to drought
stress there is less uptake of nitrate from the soil, which ultimately affect biochemical
activities like nitrate reductase (NR) (Caravaca et al., 2005) and enhances ethylene
production, which is harmful for growth and development of plant (Ali et al., 2014).

2.4. VARIOUS DEFENSE MECHANISM TO OVERCOME STRESS IN

PLANTS
i. Activation of antioxidant defense system

In order to overcome the effects of oxidative stress, plants develop self-defense

mechanisms by producing enzymatic and non-enzymatic antioxidant defense
systems that prevent ROS accumulation and alleviate the oxidative damage
occurring due to drought stress (Miller et al., 2010). Enzymatic components
include superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX),
and glutathione reductase (GR) whereas non-enzymatic antioxidants include major

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 Chapter 2 Review of Literature
cellular redox buffers, carotenoids, flavonoids, tocopherols, ascorbate and
glutathione (Apel and Hirt, 2004).
ii. Osmolytes in imparting drought tolerance in plants

Drought adaptation in plants is associated with metabolic adjustments that lead to

the accumulation of several compatible solute like proline, betaines, sugars,
polyamines, quaternary ammonium compounds, polyhydric alcohols and other
amino acids and water stress proteins like dehydrins. Proline accumulation in the
cell protects the plant by adjusting osmotic pressure as well as by stabilizing many
functional units like complex II of the electron transport system, proteins and
enzymes (Ashraf and Foolad, 2007).
iii. Plants synthesize antimicrobial phytoalexins, hypersensitive reactions, hydrolytic
enzymes and defense barriers through gelatinous materials like lignin and suberin
to protect themselves from pathogens attack (Teuber et al., 2010) and also develop
certain other self defense mechanisms like accumulation of secondary metabolites
and synthesis of defense proteins (Ashry and Mohamed, 2012). The enzymes like
peroxidases play an important role in lignification and suberization
(Hiraga et al., 2001). Recent studies indicate that microorganisms can also help
plants to cope with drought stress.

2.5. DROUGHT INDUCTION THROUGH POLYETHYLENE GLYCOL (PEG)

Field experiments related to water stress has been difficult to handle due to significant
environmental or drought interactions with other abiotic stresses (Rauf, 2008). An
alternative approach is to induce water stress through PEG solutions (Rajendran et al.,
2011; Zhang et al., 2011; Patade et al., 2012; Khodarahmpour, 2013). PEG molecules
with a molecular weight 6,000 (PEG 6000) are inert, non-ionic and cell impermeable.
Therefore, they are frequently used to simulate osmotic stress, as this solution
interferes with the roots to absorb water due to reduction of osmotic potential
(Bhargava and Paranjpe, 2004; Van den Berg and Zeng, 2006; Radhouane, 2007;
Sidari et al., 2008). To understand drought stress tolerance in plants different
morphological, physiological and biochemical parameters were investigated by many
researchers under drought stress induced by PEG (Li and Wang, 2001; Kocheva and
Georgiev, 2003; Gui-fang, 2008; Patade et al., 2012; Ye et al., 2013; Khodarahmpour,
2013; Uzilday et al., 2014). Water deficit has been known to induce a sequence of
morphological, biochemical and molecular alterations that negatively affect plant

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growth and productivity (Wu et al., 2008; Efeoglu et al., 2009). Roots are the place
where plants first encounter water stress, it is likely that roots may be able to sense
and respond to the stress condition (Xiong et al., 2006; Khodarahmpour, 2013). It
plays an important role in water stress tolerance by reduction in leaf expansion and
promotion of root growth (Achten et al., 2010). Root length at seedling stage provides
a fair estimate about the root growth in field (Ali and Ashraf, 2011; Rajendran et al.,
2011). Reduction in shoot length perhaps is due to less water absorption and decrease
in external osmotic potential created by PEG (Kaydan and Yagmur, 2008). PEG
induced water-deficit stress caused decreased percent seed germination in Niger
(Guizotia abyssinica), which is due to decreased water potential (Ghane et al., 2012).
PEG also affected seed germination by lowering its water potential in Glycine max
and Anabasis aphylla (Sakthivelu et al., 2008; Soleimani et al., 2011). Okcu et al.
(2005) reported a decrease in seed germination of Pisum sativum L. by increase in
water-deficit stress induced by PEG. Bhattacharya and Puri (2012) reported a decrease
in seed germination of Dioscorea deltoidea with an increase in concentration of PEG.

2.6. PLANT GROWTH-PROMOTING BACTERIA (PGPB)

Plant beneficial soil bacteria, regardless of where they are primarily found on the
plant, are referred to as PGPB (Glick, 2014). Bacteria enhances plant growth (Figure
2.2) either by binding to the roots (the rhizosphere) or the leaves (the phyllosphere) or
by inhabiting the interior surfaces of the plant by forming an endophytic association
(Sturz et al., 2000; Glick, 2014). The endophytic bacteria either localized in a
particular part such as roots and stem or may be distributed through-out the plant’s
tissues. The endophytes may also form nodule like specific structures, depending
upon the bacterium and the plant.

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Figure 2.2. Various plant growth promoting bacterial interactions with plants in
a range of different ways (Glick, 2014).

2.7. PLANT GROWTH PROMOTING RHIZOBACTERIA (PGPR)

PGPR are symbiotically harbored microbes on or around the root surface which
directly or indirectly affect the growth and development of plants through secreting
various chemicals in the vicinity of rhizosphere. Due to the production of growth
regulators PGPR enhances plant growth and improve plant nutrition by supplying and
facilitating nutrient uptake from soil (Zahir et al., 2004). Ahemad and Malik (2011)
stated that PGPR protect plants from phyto-pathogens and improve soil structure by
sequestering toxic heavy metals (Figure 2.3). In addition, many of the rhizobacterial
strains improve plant tolerance against salinity, drought, flooding, heavy metal
toxicity, and enable plants to survive under unfavourable environmental conditions
(Glick, 2010; Ma et al., 2011; Sandhya et al., 2009).
First examination on PGPR was done back in 1950s, since then number of
investigations on PGPR had been conducted and many applicant PGPR strains had
been screened and assessed world wide in the laboratory, greenhouse and field
conditions (Zehnder et al., 2001). These micro-organisms are vital components of
soils. They are engaged with different biotic exercises of the soil environment to make
it dynamic for supplement turn over and sustainable for crop production (Ahmad et
al., 2008).

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Figure 2.3. Mechanism of plant growth promotion by rhizobacteria (Ahemad

and Kibret, 2014)

2.7.1 Endophytic Nature of PGPR

Some bacteria and fungi enter the plant as endophytes without causing any harm to
plant and establish a mutualistic association with the host (Azevedo et al., 2000).
Dudeja et al. (2012) described the term endophyte as microbes living inside plant
tissues, without causing any disease symptoms to the plant. Endophytic bacteria are
generally found inside the roots, leaves, stems, seeds, fruits, tubers, ovules, and
legume nodules as described by Benhizia et al. (2004). Rosenblueth and Martinez
(2004) reported that in most plants, roots contain more endophytes as compared to
other aboveground tissues. For establishment endophytes must be compatible and able
to colonize host plant tissues without causing any harm to plant (Dudeja et al., 2012).
Endophytes were reported to promote seedling emergence and improve plant growth
under adverse conditions by nitrogen fixation, p-solubilization, iron chelation, acting
as antimicrobial agents, and by establishing the plant’s systemic resistance. PGPR
increases plants growth by producting phytohormones such as auxin, cytokinin, or by
producing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (Dudeja
et al., 2012). Endophytic bacteria that form nodules on specific host plants have been
known as symbiotic bacteria and have been studied extensively (Oldroyd et al., 2011;
Wang et al., 2012).

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2.7.2 Mechanism of Plant Growth Promotion by PGPR
Many microbial populations were reported as beneficial for plant growth including
bacteria, which colonize the rhizosphere and confer a positive influence on the growth
of plants (Loper et al., 2008). They influence the plant growth by direct and indirect
mechanisms as described below:

[Link] Direct mechanism

Plant root system helps the plants to find nutrients from soil for sustainable growth
and development (Zhang et al., 2012; Singh et al., 2013; Kumar et al., 2013;
Ahmad et al., 2008) (Figure 2.3). The direct mechanisms include:
(i) Phosphate solubilization
(ii) Phystohormone production
(iii) Biological nitrogen fixation

(i) Phosphate solubilization

Phosphorus (P) is the principle macronutrient for normal growth and development of
plants. Although, soil contain a large amount of P, but plants can avail only a small
proportion of it. Ramaekers et al. (2010) reported that plants only absorb the mono
and dibasic phosphate. Organic or insoluble forms of P need to be mineralized or
solubilized. To overcome the problem, farmers are applying P in the form of chemical
fertilizers. However, depending upon the soil salinity and pH, it is converted into
insoluble mineral complexes. Plants are unable to absorb these precipitated forms of
P. Here, PGPR plays a crucial role in P-solubilization. In most bacteria, mineral
phosphate-dissolving capacity is related to the production of organic acid (Devi et al.,
2015). In Gram-negative bacteria, direct oxidation of glucose to gluconic acid was
reported to be a major mechanism for mineral P-solubilisation. Furthermore, the
production of GA might significantly change the rhizosphere pH (Werra et al., 2009).
Taktek (2015) observed Burkholderia genus as efficient phosphate solubilizer.
Bacteria belonging to the genus Pseudomonas had been shown to be excellent
phosphate solubilizers that interact positively with AMF (Ordoñez et al., 2016).
Leclercia adecarboxylata and Pseudomonas putida were also reported as phosphate
solubilizing bacteria (Teng et al., 2019). Sharma et al. (2020) reported Pseudomonas
fluorescens, Pseudomonas putida, Pseudomonas koreensis and Pseudomonas
fuscovaginae and Ochrobactrum anthropic as phosphate solubilizers.

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(ii) Phytohormones production
Some of the rhizospheric bacteria were reported to synthesize phytohormones
(Lambrecht et al., 2000). The synthesis of phytohormones by bacteria often depends
on the presence of their precursors in the root exudates of plants. PGPR synthesize
and export phytohormones that are called plant growth regulators (PGR’s) (Naik et
al., 2008) which plays an important role in plant growth regulation. Earlier, PGR’s
were considered organic substances that influence physiological processes of plants at
extremely low concentrations (Dobbelaere et al., 2010). Zahir et al. (2004) reported
five classes of plant growth regulators, namely (1) auxins, (2) gibberellins,
(3) cytokinins, (4) ethylene and (5) abscisic acid. In plants, IAA is the most active
auxin, so much attention is given on its role. IAA stimulate both rapid (e.g., increases
in cell elongation) and long-term (e.g., cell division and differentiation) responses in
plants. IAA is the best characterized auxin produced by many plants associated
bacteria, including PGPR (Spaepen et al., 2007). The presence of L-tryptophan is
necessary for biosynthesis of IAA. However, some bacteria could synthesise IAA
without tryptophan. Tryptophan is a metabolic precursor of IAA in almost all soil
bacteria (Lambrecht et al., 2000). In the rhizosphere, plant root exudates are main
source of tryptophan (Glick, 2014). About 80% of rhizospheric bacteria can produce
IAA. Exogenous IAA was reported to control a number of processes in plant growth
and development, which depends upon the concentration of IAA. For example, low
concentrations of IAA stimulate primary root elongation, whereas, high concentration
stimulate the formation of lateral roots and root hairs (Perrig et al., 2007; Spaepen et
al., 2007). Bacillus amyloliqifaciens and B. subtilis produce IAA that promotes plant
growth by increasing number of roots and root hairs. These benefits of PGPR were
advantageous for not only agriculture but also for environmental purposes such as
plant-mediated bioremediation (Chen et al., 2007) and re-vegetation of eroded lands.
It had been reported that bacteria belonging to the genus Pseudomonas are efficient
IAA producers (Ahemad and Khan, 2012; Dey et al., 2004; Tank and Saraf, 2009;
Sharma et al., 2020).

(iii) Biological nitrogen fixation

Nitrogen (N2) is essential for all life forms on earth. Both organic and inorganic forms
of nitrogen are needed and recycled. In the atmosphere more than 80% of N 2 is
present, which needs to be converted into NH3, by root nodulating bacteria with the

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help of nitrogenase enzyme. Biological processes contributes 65% of the nitrogen
fixation in agriculture. While much of this by means of symbiotic N 2 fixation, non-
symbiotic and associative N2 fixation (Sylvia et al., 1999). But, still we use chemical
fertilisers, which constitute the largest human interference in the nitrogen cycle. And
has prompted concerns regarding the increased emissions of nitrogen oxides, soil
acidification and water eutrophication. Thus, fixed N 2 that is provided by biological
N2 fixation is found less prone to leaching and volatilization as it is utilized in situ.
Therefore, the biological process contributes an important and sustainable input to
agriculture. Among the N2 cycle, biological nitrogen fixation played the role of
biological conversion of atmospheric dinitrogen to many forms available for the plant
and microbial growth by a variety of prokaryotic microbes (Bhattacharjee et al.,
2008). Previously, Acetobacter diazotrophicus, Azoarcus and Cyanobacteria were
reported as the free-living nitrogen fixing bacteria (Das et al., 2013; Mehmood et al.,
2018.

[Link]. Indirect mechanism

Biocontrol activities
In the rhizosphere, plant roots interact with many plant pathogenic fungi and bacteria
that also response to root exudates. The beneficial PGPR inhibits these pathogenic
bacteria and fungi by using following mechanisms:
(i) Antifungal metabolites synthesis
(ii) Siderophore production
(iii) HCN production
(iv) Protease production

(i) Antifungal metabolites synthesis

The plant diseases caused by pathogenic bacteria and fungi are responsible for more
than US $200 billion annual crop loss. Resistant plant varieties and chemicals are
used to control the plant diseases. But, still resistance does not exist against all
diseases. Meanwhile, the breeding of resistant plants was also done for many years.
Lugtenberg and Kamilova (2009) reported that the use of PGPR strains is an alternate
solution to the problem. PGPR are reported to improve plant growth by preventing the
proliferation of phytopathogens. Some PGPR produced enzymes, which cause lysis of

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fungal cells. For example, Pseudomonas stutzeri produce extracellular chitinase and
laminarinase, which lysed the mycelia of Fusarium solani (Mauch et al., 1988).
Kapsalis et al. (2007) reported that damping-off disease of cotton caused by
Pythium and Rhizoctonia was prevented by the production of secondary metabolites
by P. oryzihabitans and Xenorhabdus nematophila strains. Fluorescent pseudomonads
were reported to exhibit strong antifungal activity against R. bataticola and F.
oxysporum, which were isolated from the rice and sugarcane rhizosphere (Kumar et
al., 2002). Even, Pseudomonas strains isolated from rhizosphere of rice was able to
suppress Xanthomonas oryzae pv. oryzae and R. solani, which were reported as a
causative agent of the bacterial leaf blight and sheath blight (Rangarajan et al., 2001).
In Indo-Gangetic plains, 50-60% of P. fluorescens was recovered from the
rhizosphere and endorhizosphere of Triticum aestivum. They were antagonistic
towards Helminthosporium sativum (Gaur et al., 2004). P. putida had potential for the
biocontrol of root-rot disease complex of chickpea by showing antifungal activity
against Macrophomina phaseolina. Moreover, Pseudomonas strains were reported
effective in suppressing Phytophthora capsici in all seasons of plant growth. P.
aeruginosa Sha8 produced toxic volatile compound which reduces the growth of both
F. oxysporium and Helmithosporium sp. while, Aspergillus niger remain unaffected
(Hassanein et al., 2009), which showed that the single antifungal metabolite could not
inhibit all types of fungal pathogens. P. fluorescens and Bacillus spp. were reported
among such commercialized PGPR organisms, which possessed the antifungal
activity against a wide variety of pathogenic fungi.

(ii) Siderophore production

Soil bacteria were also reported to chelate iron (Fe), manganese (Mn), copper (Cu),
zinc (Zn), vanadium (V), molybdenum (Mo), nickel (Ni) and cobalt (Co). Some of
these elements were reported as important in the structure and function of bacterial
enzymes, coenzymes, and cofactors. Of these elements, Fe was reported particularly
in short supply because of unavailability of Fe to cells in aerobic environments due to
low solubility of Fe oxyhydroxides near neutral pH. This form of iron could be
chelated by siderophores. Siderophore (Greek meaning “iron bearer”) denotes a
virtually Fe3+-specific ligand that was reported to produce by aerobic bacteria in
response to iron limitation with extremely diverse structure (Crosa and Walsh, 2002;
Cornelis and Matthijs, 2002). It helped in the indirect plant growth promotion that

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included the prevention of deleterious effects of phytopathogenic organisms as stated
by Dobbelaere et al. (2010). Siderophore production enabled bacteria to compete with
pathogens by removing iron from the environment for example; P. fluorescens was
observed to suppress soil-borne fungal pathogens by producing antifungal metabolites
and by sequestering iron in the rhizosphere through siderophores, and thus rendering
it unavailable to other organisms (Dwivedi and Johri, 2003). Miethke et al. (2006)
reported Bacillibactin, a Bacillus-produced siderophore and classified it as an
antibiotic. This siderophore had been reported to be produced by different strains of
B. subtilis. It was found to be expressed in the PGPR strain B. amyloliquefasciens
FZB42 that improved plant growth (Chen et al., 2007). Acinetobacter calcoaceticus
showed siderophore mediated antagonism against common phytopathogens like, A.
flavus, A. niger, Colletotrichum capsicum and F. oxysporum (Prashant et al., 2014).
Bacteria belonging to genus Pseudomonas and genus Ochrobactrum were reported as
efficient siderophore producers by Sharma et al., 2020.

(iii) HCN production

Cyanide was accounted as a secondary metabolite produced by some
gram negative bacteria, for example P. fluorescens, P. aeruginosa, and
Chromobacterium violaceum (Chen and Kuo, 1993). It was reported to be
synthesized, discharged and utilized by many life forms, including microbes, algae,
fungi, plants and insects, as an intend to avoid predation or competition. Earlier,
Castric (1977) reported the production of HCN and CO 2 stoichiometrically from
glycine. Further studies revealed that glycine is a precursor for HCN production in
P. aeruginosa. Ahmad et al. (2008) and Charest et al. (2005) reported HCN
production as a typical characteristic of Pseudomonas (88.89%) and Bacillus (50%),
which were available in the rhizospheric soil and plant root nodules.
Schnider-Keel et al. (2001) reported P. fluorescens as a root-colonizing
biocontrol bacterium, which protects several plants from root diseases caused by soil
borne fungi. Wani et al. (2007) reported that most of the rhizospheric isolates are able
to produce HCN and helpful in the plant growth. The isolates from the rhizospheric
soil of chickpea were observed to exhibit PGPR traits along with HCN production,
which promoted plant growth directly or indirectly or synergistically (Joseph et al.,
2007). Under normal growth conditions Mesorhizobium loti produced HCN and
enhanced the growth of Brassica campestris (Chandra et al., 2007). The P. fragi, a

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Chapter 2 Review of Literature
psychro tolerant bacterium was reported to produce HCN and the seed bacterization
with the isolate significantly increased the rate of germination, percentage
germination, plant biomass and nutrient uptake of Triticum aestivum seedlings
(Selvakumar et al., 2009).
Interestingly, the cyanide-producing bacterial strains did not affect the host
plant and host-specific rhizobacteria acted as biological weed-control agents
(Zeller et al., 2007). Even, some plants like Lima bean (Phaseolus lunatus L) were
reported to release hydrogen cyanide in response to damage caused by natural
enemies, thereby directly defending plant tissue (Ballhorn et al., 2008).

2.7.3 Role of PGPR to Overcome Drought Stress

PGPR play a significant role in agriculture to promote the exchange of nutrients and
reduce use of fertilizers. The plant-microorganism collaborations in the rhizosphere
affectes plant life and soil fertility by stimulating plant growth through mobilizing
nutrients in soil, producing various growth hormones by controlling or hindering
phytopathogens, improving soil structure and bio-remediating the contaminated soils
by sequestering lethal heavy metals and degrading pesticides (Bhattacharyya and Jha,
2012).

[Link] Activation of osmolytes due to inoculation of PGPR

In response to drought stress, PGPR secrete osmolytes, which acts synergistically

with plant-produced osmolytes and regulate plant growth (Paul et al., 2008).
Inoculation of P. putida improved plant biomass, relative water content and leaf water
potential by accumulation of proline in maize plants exposed to drought stress
(Sandhya et al., 2010). Inoculating plants with PGPR enhanced concentrations of
proline, when maize plants were inoculated with P. fluorescens under drought stress
(Ansary et al., 2012). Increased proline level in plants maintain cell water status,
protects cell membranes and proteins from stress (Sandhya et al., 2010). Similarly,
Shintu and Jayaram (2015) reported that tomato (Lycopersicon esculentum Mill)
treated with B. polymyxa secreted excess proline to mitigate the drought condition.
Higher proline accumulation in inoculated plants indicate higher water stress
tolerance in plants (Gusain et al., 2015). Under drought stress inoculation of maize
plant with PGPR P. putida (Sandhya et al., 2010) and A. lipoferum (Bano et al., 2013)
improved plant growth through accumulation of free amino acids and soluble sugars.

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Trehalose, a non-reducing disaccharide stabilizes dehydrated enzymes and
membranes; and acts as osmoprotectant (Yang et al., 2010). Paul et al. (2008)
described that trehalose plays an important role as a signaling molecule in plants.
Choline plays an important role in plant stress resistance by enhancing synthesis and
accumulation of glycine betaine (Zeisel, 2006; Zhang et al., 2010). Evident reports on
induced role of B. subtilis in Arabidopsis (Zhang et al., 2010) and Klebsiella
variicola, P. fluorescens and Raoultella planticola in maize showed enhancements in
biosynthesis and accumulation of choline (precursor of glycine betaine) resulting in
the accumulation of glycine betaine thereby improving leaf relative water content
(RWC) and dry weight (Glick et al., 2007; Zhang et al., 2010; Gou et al., 2015).

The increased content of choline in drought stressed maize plants supplied

more nutrition as a food additive (Zeisel, 2006; Zhang et al., 2010). Under drought
stress condition, PGPR enhances accumulation of solutes such as glycine betaine that
regulate plant stress responses by preventing water loss caused by osmotic stress
(Nadeem et al., 2010; Bashan et al., 2014). Inoculation of B. subtilis and
Pseudomonas spp. in osmotic stressed plants resulted in high accumulation of glycine
betaine than un-inoculated plants (Sandhya et al., 2010). Similarly, polyamines are
considered as plant growth regulating compounds and cadaverine is correlated with
root growth promotion in plants. Cadaverine producing A. brasilense promoted root
growth and alleviated osmotic stress in rice seedlings (Cassan et al., 2009).

Table 2.1. PGPR enhances osmolytes accumulation in imparting drought

tolerance in plants.

Plant
PGPR Effect Reference
species
Pseudomonas putida Zea mays Accumulation of proline Sandhya
GAP-P45 improved plant biomass, RWC et al.,
and leaf water potential 2010
Pseudomonas Zea mays Increased proline, abscisic acid, Ansary et
fluorescens auxin, gibberelline and al., 2012
cytokinin content improved
plant growth
Bacillus thuringiensis Lavandula IAA induced higher proline and Armada et
dentate K-content, improved al., 2014
nutritional, physiological, and
metabolic activities and
decreased GR and APX activity
Bacillus polymyxa Lycopersicon Proline accumulation improved Shintu
esculentum the physiological and and

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Chapter 2 Review of Literature
biochemical parameters of Jayaram,
plants 2015
Consortia containing Oryza sativa Accumulation of proline Gusain et
Pseudomonas jessenii, maintained osmotic adjustment al., 2015
R62, Pseudomonas and improved plant growth
synxantha, R81 and
Arthrobacter
nitroguajacolicus
strainYB3, strain YB5
Azospirillum lipoferum Zea mays Improved plant growth through Bano et
accumulation of free amino al., 2013
acids and soluble sugars
Azospirillum Zea mays Trehalose translocated to the Rodriguez
brasilense maize roots and triggered stress et al.,
overexpressing tolerance pathways in the plant 2009
trehalose biosynthesis
gene
Bacillus subtilis GB03 Arabidopsis Expression of PEAMT gene Zhang et
resulted in elevated metabolic al., 2010
level of choline together with
GB in osmotically stressed
plants and improved leaf RWC
and dry DMW
Klebsiella variicola F2, Zea mays Accumulation of choline and Gou et al.,
Pseudomonas GB and improved leaf RWC 2015
fluorescens YX2 and and DMW
Raoultella planticola
YL2
Azospirillum Oryza sativa
Cadaverine production by Cassan et
brasilense Az39 PGPR improved root growth al., 2009
and mitigated osmotic stress
Key: K=Potassium; RWC=Relative water content; IAA=Indole acetic acid;
GR=Glutathione reductase; APX=Ascorbate peroxidase; DMW=Dry matter
weight; GB=Glycine betaine; PGPR=Plant growth promoting rhizobacteria

2.7.4 Mechanisms of PGPR Mediated Drought Stress Tolerance

Several mechanisms like phyto-hormonal activity, volatile compounds, alteration in
root morphology, ACC deaminase activity, accumulation of osmolytes, EPS
production, antioxidant defense and co-inoculations have been proposed for PGPR
mediated drought stress tolerance in plants.

[Link]. Modification of phytohormonal activity in imparting drought tolerance

in plants
Various phytohormones such as IAA, gibberellins, ethylene, abscisic acid and
cytokinins produced by plants play an important role in their growth and development
(Teale et al., 2006; Egamberdieva, 2013). These phytohormones help the plants to

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Chapter 2 Review of Literature
escape or survive under unfavourable conditions (Skirycz and Inze, 2010; Fahad et
al., 2015). Earlier Glick and Pasternak (2003) reported that PGPRs synthesize
phytohormones that stimulate plant cell growth and division to become tolerant
against environmental stresses. In various plant species inoculation of IAA producing
bacteria enhanced root growth and formation of lateral roots and roots hairs (Dimkpa
et al., 2009), increasing water and nutrient uptake (Mantelin and Touraine, 2004),
helping plants to tolerate drought stress (Egamberdieva and Kucharova, 2009). IAA
producing Azospirillum enhanced plant’s tolerance to drought stress
(Dimkpa et al., 2009). A. brasilense producing nitric oxide, act as a signaling
molecule in IAA induction pathway and helps in formation of adventitious roots in
tomato plants (Creus et al., 2005; Molina-Favero et al., 2008). Inoculation of maize
seedlings with A. brasilense improved relative and absolute water content as
compared to un-inoculated plants under drought stress. Bacterial treatment dropped
the water potential and increased the root growth, biomass, foliar area and proline
accumulation in leaves and roots. Azospirillum-Triticum aestivum association induced
decrease in leaf water potential and increase in leaf water content, which was
attributed to the production of IAA by the bacteria that enhanced root growth and
formation of lateral roots and increases uptake of water and nutrients under drought
stress (Arzanesh et al., 2011). B. thuringiensis produced IAA, which improved
nutrition, physiology, and metabolic activities of Lavandula dentate plants (Armada et
al., 2014). Glycine max plants inoculated with P. putida secrets gibberellin, which
improved plant growth under drought conditions (Sang-Mo et al., 2014). Production
of ABA and gibberellins by A. lipoferum decreased drought stress in maize plants
(Cohen et al., 2009). During drought condition, cellular dehydration induces
biosynthesis of a stress hormone Abscisic acid (ABA) (Kaushal and Wani, 2016).
ABA is involved in water loss regulation by controlling stomatal closure and stress
signal transduction pathways. Arabidopsis plants inoculated with A. brasilense
showed high ABA concentration as compared to un-inoculated plants (Cohen et al.,
2008). Inoculation of Phyllobacterium brassicacearum, elevating ABA content in
Arabidopsis plants leading to decreased leaf transpiration and enhanced osmotic stress
tolerance (Bresson et al., 2013).

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Chapter 2 Review of Literature
Table 2.2. PGPR phytohormonal activity in imparting drought tolerance in
plants
Plant
PGPR Effect Reference
species
Azospirillum Lycopersic Nitric oxide as a signaling Creus et al.,
brasilense on molecule in IAA induced 2005; Molina-
esculentum pathway which enhanced Favero et al.,
lateral root and root hair 2008
development
Azospirillum Zea mays Gibberellins increased ABA Cohen et al.,
lipoferum levels and alleviated drought 2009
stress
Azospirillum sp. Triticum IAA enhanced root growth, Arzanesh et al.,
aestivum lateral roots formation and 2011
increased uptake of water and
nutrients under drought stress
Phyllobacterium Arabidopsi Enhanced ABA content Bresson et al.,
brassicacearum s resulted in decreased leaf 2013
strain STM196 transpiration
Bacillus subtilis Platycladus Cytokinin production by Liu et al., 2013
orientalis PGPR elevated ABA levels in
shoots and increased the
stomatal conductance
Pseudomonas Glycine Secretion of gibberellins by P. Sang-Mo et al.,
putida H-2–3 max putida improved plant growth 2014
Bacillus Lavandula IAA resulted in higher K- Armada et al.,
thuringiensis dentate content, proline and decreased 2014
the glutathione reductase (GR)
and ascorbate peroxidase
(APX)
Rhizobium Triticum IAA produced by the
Hussain et al.,
leguminosarum aestivum consortia improved 2014the
(LR-30), growth, biomass and drought
Mesorhizobium tolerance index
ciceri (CR-30 and
CR-39), and
Rhizobium
phaseoli (MR-2)
Key: IAA=Indole acetic acid; ABA=Abscisic acid; GR=Glutathione reductase;
PGPR=Plant growth promoting rhizobacteria; K=Potassium; APX=Ascorbate
peroxidase

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Chapter 2 Review of Literature

Figure 2.4. fvMechanism of rhizosphere microbes to minimize the various types of abiotic and biotic stress for enhancement of
plant growth and development, ROS: Reactive Oxygen Species; ABA: Abscisic Acid; EPS: Exopolysaccharide; ACC:
1- Aminocycloprpane-1-Carboxylate; AMF: Arbuscular Mycorrhizal Fungi; Pb: Lead; Cr: Chromium. (Kumar and
Verma, 2018)

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Chapter 2 Review of Literature

Figure 2.5. Mechanism of plant drought tolerance induced by plant growth promoting rhizobacteria (Vurukonda et al., 2016)

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Chapter 2 Review of Literature

[Link] Exopolysaccharide (EPS) production by rhizobacteria and alleviation of

drought stress

EPSs are sticky organic macromolecules with a high molecular weight. Bacterial
EPSs are chemically diverse molecules and play an important role in the interaction
between bacteria and their environment (Almansoory et al., 2020). The EPS are
capsular and slime materials released by soil microbes into the soil and can be
adsorbed by clay surfaces due to cation bridges, hydrogen bonding, Van der Waals
forces and anion adsorption mechanisms thus forming a protective capsule around soil
aggregates (Sandhya et al., 2009). EPS provides a microenvironment that holds water
and dries up more slowly than the surrounding environment thus protect bacteria and
plant roots against desiccation. Bacterial EPS improve permeability by increasing soil
aggregation and maintaining higher water potential around the roots, thereby
increases nutrients uptake by plant and increases plant growth and protect them from
drought stress (Alami et al., 2000; Selvakumar et al., 2012). Plants treated with EPS-
producing bacteria show resistance against drought stress. Exopolysaccharides play an
important role in plants exposed to water deficit conditions. As drought affect plants
and microbial population, the exopolysaccharides protect the bacteria and plants from
desiccation, and enable them to continue their growth under dry condition (Sandhya et
al., 2009). Drought stress alters physico–chemical and biological properties of soil
and makes it unfit for soil micro-organisms and crop yield. EPS produced by
microbes protect them from unfavourable conditions and ensure their survival.
Capsular material of A. brasilense contain high molecular weight carbohydrate
complexes, which protect it under extreme desiccation conditions. Addition of these
complexes to a suspension of decapsulated cells of A. brasilense significantly
enhances survival under drought stress (Konnova et al., 2001). Seed bacterization of
maize with EPS-producing bacterial strains Proteus penneri, P. aeruginosa and
Alcaligenes faecalis along with the irrespective EPS improved soil-moisture content,
plant biomass, root and shoot length and leaf area. Under drought stress, the
inoculated plants showed increase in relative water content, protein, sugar and proline
content and decrease in the activities of antioxidant enzymes (Naseem and Bano,
2014).

[Link]. PGPR alter root morphology under drought stress

Plant’s physiological status is regulated by cell membranes and rhizobacteria can

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Chapter 2 Review of Literature
influence processes taking place at these sites. Pereyra et al., 2006 reported that A.
brasilense reduces Triticum aestivum seedlings membrane potential and phospholipid
content in the cell membranes of cow-pea due to the changes in proton efflux
activities. Drought changes the phospholipid composition in the root, increases
phosphatidyl choline and reduces phosphatidyl ethanolamine, but inoculation with
Azospirillum prevented these changes in Triticum aestivum seedlings, though higher
phosphatidyl choline and lower phosphatidyl ethanolamineun saturation occurred
(Pereyra et al., 2006). Bacteria mediated changes in the elasticity of the root cell
membranes is one of the first step towards drought resistance (Dimkpa et al., 2009).
PGPR activate the antioxidant defense system and improves the stability of plant cell
membranes and enhances drought tolerance in plants (Gusain et al., 2015).

[Link] Role of ACC deaminase producing rhizobacteria in drought stress

tolerance

The ACC deaminase is a pyridoxal 50-phosphate dependent enzyme that catalyses the
conversion of ACC to ammonia and α-ketobutyrate (Yao et al., 2000). The diminution
of ethylene concentration via microbial deamination of ACC was identified as another
phyto-beneficial mode of action exhibited by both PGPR and nitrogen-fixing bacteria
(Holguin and Glick, 2003). Ethylene was required for the induction of systemic
resistance in plants during associative and symbiotic plant-bacteria interactions and at
higher concentrations it was involved in plant defence pathways induced in response
to pathogen infection (Broekaert et al., 2006: Glick et al., 2007). Although ethylene is
essential for the normal growth and development in plants, but at high concentration it
is harmful as it induces defoliation and other cellular processes that may lead to
reduction in crop yield. Ethylene was reported as a key phytohormone that can inhibit
the root elongation, nodulation, auxin transport and promotes seed germination.
Similarly, senescence, abscission of various organs and fruit ripening was also
reported to be promoted by ethylene (Glick et al., 2007). Rhizobacteria assisted in
diminishing the accumulation of ethylene levels and re-establish a healthy root system
that was observed as an essential factor to cope with the environmental stress. In this
process primary mechanism included the destruction of ethylene by the activity of
enzyme ACC deaminase. This ACC deaminase activity was found in many
rhizobacteria such as Achromobacter, Azospirillum, Bacillus, Enterobacter,
Pseudomonas and Rhizobium (Ghosh et al., 2003). Certain PGPR such as

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Chapter 2 Review of Literature
A. brasilense had been shown to produce small amounts of ethylene from methionine
as a precursor (Perrig et al., 2007) and this ability promoted root hair development in
tomato plants (Ribaudo et al., 2006). As less ethylene is synthesized, the inhibitory
effects on root elongation also decreases. The reaction products of ACC hydrolysis
are used as nutrients for growth by PGPR. It is reported that plants inoculated with
ACC deaminase-positive PGPR strain reduces ethylene levels in the root zone and
hence increases root proliferation. Mayak et al. (2004) observed a significant increase
in fresh and dry weight of tomato plants inoculated with Achromobacter piechaudii
under water and saline stress conditions.
Plant activities are regulated by ethylene levels and ethylene synthesis is
regulated by biotic and abiotic stresses (Hardoim et al., 2008). ACC is the immediate
precursor of ethylene. Under stress conditions, ethylene regulates plant homoeostasis
resulting in reduced root and shoot growth. ACC deaminase producing bacteria
degrade the ACC to supply nitrogen and energy to plant and hence reduces the
harmful effect of excess ethylene to plant, ultimately reduces plant stress and helps in
promoting plant growth (Glick, 2005). Under drought stress, ACC deaminase
producing Achromobacter piechaudii increased the fresh and dry weight of tomato
and pepper seedlings and decreased the ethylene production (Mayak et al., 2004).
Inoculation of ACC deaminase producing bacteria in plants resulted in more seed
yield, seed number, seed nitrogen accumulation and root nodulation, which was low
in drought stress (Dodd et al., 2005).

Table 2.3. ACC deaminase producing PGPR in mitigation of drought stress in

plants

PGPR Plant Effect Reference

species
Achromobacter Lycopersicon Reduced ethylene production Mayak et al.,
piechaudii ARV8 esculentum and increased fresh and dry 2004
and weight
Capsicum
annuum
Variovorax Pisum Increased nodulation, seed Dodd et al.,
paradoxus 5C-2 sativum yield, seed number and seed 2005
nitrogen content
Pseudomonas Pisum Induced longer roots and
fluorescens sativum increased uptake of water
biotype G (ACC-
5)

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Chapter 2 Review of Literature
Variovorax Pisum Increase in xylem abscisic Belimov et al.,
paradoxus 5C-2 sativum acid, higher nodulation, 2009
growth, yield and water-use
efficiency
ACC deaminase Triticum Increased root-shoot length, Shakir et al.,
producing aestivum biomass and lateral root 2012
rhizobacteria number
Consortia of Cicer Higher proline concentration, Sharma et al.,
Bacillus isolate 23- arietinum improved germination, root 2013
B and and shoot length and fresh
Pseudomonas 6-P weight of the seedlings
with
Mesorhizobium
ciceris
Bacillus CapsicumHigher expression of stress Hui and Kim,
licheniformis K11 annuum genes Cadhn, VA, sHSP and 2013
CaPR-10
Bacillus Triticum Reduction of volatile Timmusk et al.,
thuringiensis aestivum emissions and higher 2014
AZP2 photosynthesis
Key: ACC=1-Aminocyclopropane-1-carboxylate; VA=Vacuolar H+-ATPase;
sHSP =Cytoplasmic small heat shock protein; CaPR-10=Pathogenesis- related
protein 10 (Capsicum annuum)

Arshad et al. (2008) reported the effects of ACC deaminase producing bacteria on
growth, yield and ripening of Pisum sativum in both in vitro and in vivo conditions.
Inoculation of Pisum sativum with ACC deaminase producing Pseudomonas
fluorescens resulted in enhancement of root growth due to which water uptake
increases under drought stress (Zahir et al., 2008). Better growth of roots helped
plants to acquire water and nutrients resulting in improved growth and yield under
drought stress (Shakir et al., 2012).

2.8 Spilanthes acmella Murr.

S. acmella Murr. is an important medicinal plant among the varied and rich diversity
of medicinal plants of Indian Himalayan Region (IHR). It is widely used for the
treatment of various ailments. S. acmella Murr. (Compositae) is the well-known
“toothache plant”, commonly known as Akarkara, occurring throughout India,
ascending to 1700 m. It has a long history of folklore remedy for toothache,
rheumatism and fever (Wongsawatkul et al., 2008). Asteraceae (Compositae) is the
largest vascular plant family with 30,000 sp. and over 1100 genera. Genus Spilanthes
belongs to this family and is widely distributed in tropical and sub -tropical regions of
the world. Around 60 species of genus Spilanthes have been reported from different

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Chapter 2 Review of Literature
regions of the world. Out of these, five species occur in India namely S. acmella
Murr., S. acmella L., var. oleracea Clarke, S. calva L., S. peniculta L., and S.
mauritiana L. (Tiwari et al., 2011). S. calva, S. paniculata and S. mauritiana grow
widely. S. oleracea is rare in occurrence whereas S. acmella is an acutely threatened
species. In India, it is reported from some of the regions of South India, Chhattisgarh
and Jharkhand (Tiwari et al., 2011) and in Rajasthan (Sharma et al., 2010). The plant
genus grows naturally in damp areas, near lakes or ponds and near sewage discharge
areas and also grown at Dr. Yashwant Singh Parmar University of Horticulture and
Forestry Nauni – Solan, Himachal Pradesh (Chauhan, 2006).

Figure 2.6. Plant of Spilanthes acmella Murr.

S. acmella is an annual herb, more or less pubescent, sometimes hairy. Plants are
generally around 40 to 60 cm tall. The stems are prostrate in some of the species and
erect in others, with opposite, triangular or ovate-lanceolate leaves, about
5cm×3.75cm and dentate (toothed) or almost entire margins. The flower heads are
white or yellow in color, cone- like or ovoid, long peduncles and solitary, mostly
bisexual (Chauhan, 2006). The roots are long and tapering and also emerge from the
nodal regions in prostrate plants. In some of the species, the heads have a dark red
spot in the center. The seeds are small, grey and black in color. This plant genus
grows throughout the year in tropical regions with rich soil and organic compost.
Regular watering and shady conditions are essential for the proper growth of the
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Chapter 2 Review of Literature
plant. Genus Spilanthes has no serious disease problems although it may occasionally
suffer from spider mite damage. It has poor vegetative propagation and a low rate of
germination (Rios-Chavez et al., 2003). Whole plant (roots, flower heads and aerial
part) of S. acmella yield spilanthol, which is a powerful stimulant, sialagogue and
used as local anesthetic.

Medicinal value of S. acmella Murr.

S. acmella Murr. (Asteraceae) is the well-known toothache plant, also commonly used
as a spice. It has a long history of use as a folklore remedy, e.g., for toothache,
rheumatism and in fever (Wongsawatkul et al., 2008). The plant has found
applications in pharmaceuticals as an anti-toothache formulation for pain relief
(Pandey, 2007), swelling and gum infections (Pandey, 2007), periodontosis
(Adler, 2006) and in mouthwashes. Leaves are used in bacterial and fungal skin
diseases and as salad. Roots are used as purgative and laxative, in cold and flu, in
headache and asthma. Flower heads are used to relieve infection of throat, gum and
paralysis of tongue. A tincture made from flower head is used as substituent for the
tincture of pyrethrum to treat the inflammation of jawbone and caries. It is regarded as
stimulant and sialogogue, local anesthetic and powerful insecticide. Leaves are used
in sore throat. The leaves may be used topically to treat bacterial and fungal skin
diseases such as ringworm scan. Internally, the decoction of leaves are given as
diuretic and lithotriptic. Seeds are also chewed to relieve toothache and infection of
throat, gums and paralysis of tongue. Decoction of root is used as purgative, a gargle
in dental caries, odontalgia, cholera, rheumatism and thyphus fever. Whole plant is
used as a fish poison, in dysentery and against scurvy. The herb exhibits general
immunomodulator properties when used internally, boosting production of leukocytes
and antiviral interferon, as well as promoting phagocytosis (Yoganarasimhan, 2000).
The plant has found applications in pharmaceuticals as an anti-toothache formulation
for pain relief, swelling and gum infections (Pandey et al., 2007) and in mouthwashes.
In addition, its extract is an active component added to body and beauty care
cosmetics as a fast-acting muscle relaxant to accelerate repair of functional wrinkles
(Belfer, 2007). S. acmella Murr. also exhibits vasorelaxant and antioxidant activities
(Wongsawatkul et al., 2008). In Ayurvedic system of medicine, flower heads and
roots are used in treatment of scabies, psoriasis, scurvy, toothache, infections of gums
and throat, paralysis of tongue and remedy for stammering in children. Because of its

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high medicinal value, there is a much demand of this plant in the global market. It has
been reported to show antibacterial, antifungal, and antimalarial activity.
Traditionally, this plant is used in treatment of toothache, flue, cough, and
tuberculosis (Tiwari et al., 2011). The antimicrobial activity is mainly due to the
presence of an alkaloid spilanthol (N-isobutyl-2, 6, 8 Decatrienamide)
(Saritha et al., 2002; Manukyan, 2011). Spilanthol has a strong pungent taste; it may
produce local astringency and anesthetic effects (Chakraborty et al., 2002;
Senthilkumar et al., 2007).

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