Chapter 1 Introduction

Drought is one of the leading causes of reducing food production in the world over
(Vinocur and Altman, 2005; Naveed et al., 2014). Due to differences in
hydro-meteorological variables, socio-economic factors and the stochastic nature of
water demands in different regions of the world, various definitions of drought have
been suggested (American Meteorological Society, 2004; Ngumbi and
Kloepper, 2016). Depending on the variable used to define drought, its definitions are
divided into four categories: (1) meteorological drought, characterized as an absence
of precipitation for a while (2) hydrological drought, defined as a lack of sufficient
surface and subsurface water resources for established water uses (3) socio-economic
drought, characterized as the failure of water assets frameworks to fulfill water
demands and (4) agricultural drought, defined as a period with declining soil moisture
bringing about crop failure (American Meteorological Society, 2004). In plants
drought is generally referred when there is scarcity of water during growth period that
disturbs physiological functions, which result in growth reduction and no production.
Indeed, at some time, most of the land plants in their life cycle are exposed to short
or long term water stress and have tended to develop some adaptive mechanisms for
changing environmental conditions. Water stress may range from moderate, to
extremely severe and prolonged summer drought which strongly influences evolution
and plant life. Water deficit is a major abiotic stress factor resulting in reduced global
crop yield (Manavalan et al., 2009). It is important to state that different plant species
are highly variable with respect to their optimum environment, particularly for
requirement of water.
Response to drought stress occurs at all levels of plant organization i.e., at
cellular structure physiological functions, genetic level etc. Despite it, during
insufficient water conditions, plants experience a progression of physiological and
molecular change, for example, increase in ethylene production, change in
chlorophyll content, harm photosynthetic mechanical assembly and hinder
photosynthesis (Lata and Prasad, 2011). Constrained water content lessens cell size,
membrane integrity, produces reactive oxygen species (ROS) and promotes leaf
senescence that leads to diminished crop production (Tiwari et al., 2016). Drought
stress also influences plants in terms of hormone composition, protein changes,
osmotic adjustment, antioxidant production, root depth and extension, inhibition of
photosynthesis, opening and closing of stomata, cuticle thickness, decrease in
chlorophyll content, reduction in transpiration and growth inhibition to stand with
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Chapter 1 Introduction
some osmotic changes in their organs (Zhu, 2002; Lawlor and Cornic, 2002;
Yordanov et al., 2000). Likewise, drought stress brings about the accumulation of free
radicals that induce a change in membrane function, protein conformation, lipid
peroxidation and, lastly, cell demise (Tiwari et al., 2016). Drought stress can cause
the formation of ROS and the alteration of water potential within the plant
(Sanchez-Rodriguez et al., 2010). The ROS is harmful to cell membranes, resulting in
oxidative degradation of membrane lipids. Water deficit has been known to induce
morphological, biochemical and molecular alterations that reduces plant growth and
crop yield (Wu et al., 2008; Efeoglu et al., 2009).
The ability of plants to sustain normal growth under drought stress and survive
during unfavourable conditions has been termed as drought resistance
(Chaves et al., 2003). Several mechanisms can be employed to mitigate the drought
stress like morphological alterations, tuning of osmolytes, augmentation of diverse
water resources, modification of antioxidant defense systems to reduce ROS related
changes and, reorientation of stress-related genes (Farooq et al., 2009). Other methods
include improving the breed of plants either by traditional methods or by transgenic
approach and development of new irrigation techniques. However, these methods are
highly labour-intensive, time consuming and often tough to apply in field conditions
(Niu et al., 2018).
Vinocur and Altman (2005) stated, by 2050 almost 50% of the earth’s
cultivable land would get affected due to low irrigation, leading to severe decrease in
the agricultural produce. Simultaneously, world population is forecasted to be around
9 billion by 2050 and expected to face massive gap in demand and supply chain
(Gatehouse et al., 2011). This calls for an increase in crop production. Therefore, it is
a dire need to find solutions to abate the consequences of drought on agriculture
productivity (Alexanratos and Bruinsma, 2012). Particularly, there is a need to find
solutions to increase plants’ tolerance against drought stress and allow the crops to
grow and fulfill the food demand under limited water resources (Mancosu et al.,
2015).
However, it is not an easy task and would require different strategies and
approaches to tackle this issue. In the present time, growers are dependent on
chemical fertilizers, insecticides and fungicides to provide food security to the world.
These synthetic compounds dominate the market and are being used excessively, thus
affecting the soil health and certain level of toxicity to the crops (Savci, 2012).
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Chapter 1 Introduction
Furthermore, the consumer concern for safer food and more ecologically compatible
plant disease management practices for sustainable agricultural development are
growing fast. New strategies like the development of bio-pesticides to suppress pest
populations (Ongena and Jacques, 2007) and the use of soil microorganisms i.e., plant
growth-promoting rhizobacteria (PGPR) in place of chemical fertilizers have
emerged. PGPR colonize the rhizosphere of many plant species giving beneficial
effects and cause physical or chemical changes related to plant defense. This enables
the plant to tolerate abiotic stresses.
Some of the plant-associated microbes, like Azotobacter, Arthrobacter,
Pseudomonas, Bacillus, Clostridium, Hydrogenophaga, Enterobacter, Serratia,
Bukholderia and Azospirillum have been identified as PGPR (Lugtenberg and
Kamilova, 2009). Using these diverse organisms, crop productivity can be increased
considerably (ICAR Report, 2006-2007). PGPR colonizes in the rhizosphere, the
narrow zone of soil immediately surrounding the root system and is much more
abundant in bacteria than the surrounding bulk soil (Adesemoye et al., 2009). The
concentration of bacteria was reported 10 to 1000 times higher in the rhizosphere as
compared to surrounding bulk soil (Lugtenberg and Kamilova, 2009). This
phenomenon, termed as rhizosphere effect, causes a substantial amount of the carbon
fixed (5–21%) by the plant, is secreted as root exudate (Armstrong et al., 2009). Watt
et al. (2006) observed microbial colonization of the plant root surface in patches
covering ~15-40% of the total plant root surface. The process of root colonization was
observed under the influence of various parameters such as bacterial traits, several
other biotic, abiotic factors and root exudates. By the secretion of root exudates, the
roots regulate the soil microbial community in the closer vicinity to cope with
herbivores, encourage beneficial symbiosis, changes the chemical and physical
properties of the soil and inhibit the growth of competing plant (Lugtenberg and
Kamilova, 2009). The root exudates play an important role as chemical attractants and
repellents in the rhizosphere (Bais et al., 2006). Mostly the bacterial colonization and
attachment were reported at the junction of the epidermal cell, root hairs, axial
grooves, cap cells and sites of emerging lateral roots (Danhorn and Fuqua, 2007).
Whipps (2001) classified plant-microbe interaction into three categories i.e., neutral,
negative and positive. Knief et al. (2011) defined most of the neutral rhizobacteria
associated with plants are commensals in which the bacteria and the host plant forms
an innocuous interaction exhibiting no visible effect on the growth and overall
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Chapter 1 Introduction
physiology of the plant. Whereas, in negative interactions, the phytopathogenic
rhizobacteria produce phytotoxic substances such as ethylene; thus, negatively
influence the growth and physiology of the plants.
Further, it was reported that to counter these unhealthy bacteria, there are
some PGPRs which exert a positive plant growth. These beneficial bacteria can
enhance the plant growth by direct and indirect mechanisms. Direct mechanism
includes nutrient solubilization, nitrogen fixation, production of growth regulators,
etc., whereas indirect mechanisms comprise stimulation of mycorrhizae development,
competitive exclusion of pathogens or removal of phytotoxic substances (Lugtenberg
and Kamilova, 2009). Direct mechanisms involve the increment in plant growth by
making the availability of nutrients to the growing plant. It is mostly done by
phosphate solubilization, indole-3 acetic acid (IAA) production, phytase production,
nitrogen fixation, siderophore production and ammonia production. Bacteria
possessing these traits could promote plant growth under a variety of stressful
conditions, such as flooding, saline and drought (Kwak et al., 2012; Perez-
Pantoja et al., 2012). Indirect mechanisms involve crop protection from the pathogens
and improve plant health. Mainly these mechanisms include the production of
antifungal compounds, hydrogen cyanide (HCN), chitinase, siderophore and protease,
which were reported to play an essential role in biocontrol activity. Zhang et al.
(2012) had reported insecticidal activity (Loper et al., 2008) and production of
germination arrest factor by PGPR strains.
Vardharajula et al. (2011) reported drought-resistant Bacillus spp. that
exhibited many plant growth promoting (PGP) properties under drought stress and
improved the overall growth of maize plants under drought stress of -0.73 MPa,
25% polyethylene glycol (PEG). Three bacterial strains Pseudomonas putida,
Pseudomonas strain GAP-P45 and Bacillus megaterium isolated from drought soil
were able to encourage plant growth under drought conditions (Marulanda et al.,
2009; Sandhya et al., 2009). In drought-stress environment, the inoculation of PGPR
is the most effective method to increase productivity. PGPR produce phytohormones,
which ultimately enhance the plant growth. Production of IAA, an active auxin by
several microorganisms including PGPR through the L-tryptophan metabolic pathway
helps plant to grow (Marulanda et al. 2009; Van et al., 2000; Ciccillo et al., 2002).
Under osmotic stress conditions, production of IAA by PGPR was considered as a
suitable marker for its effectiveness to overcome the drought stress (Boiero et al.,
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Chapter 1 Introduction
2006). Similar studies were done to analyse the impact of bacteria on plant growth,
Burkholderia phytofirmans strain PsJN and Enterobacter sp. strain FD17 were
reported to improve the overall growth of plant in drought stress (Ngumbi, 2011;
Naveed et al., 2014; Humaira et al., 2013; Vardharajula et al., 2011; Sandhya et al.,
2010; Naseem and Bano, 2014). Similarly, under drought stress, an increase in plant
growth due to PGPR treatment have also been reported in other crops like Vigna
radiata L., Helianthus annuus L., Sorghum bicolor and Triticum aestivum
(Saravanakumar et al., 2011; Sarma and Saikia, 2014; Castillo et al., 2013; Grover et
al., 2014; Arzanesh et al., 2011; Kasim et al., 2013; Sandhya et al., 2009).
Certain PGPR strains can produce exopolysaccharides (EPS) that play an
essential role in plants exposed to drought stress conditions. Bacterial EPS are
hydrated compounds with 97% of the water in a polymer matrix, which protect
against aridity (Hunter and Beveridge, 2005; Bhaskar and Bhosle, 2005). The EPS
protect these bacteria from desiccation under drought stress by enhancing the water
retention. The plants treated with EPS producing bacteria, Azospirillum showed
resistance to drought stress by improving the soil structure and soil aggregation
(Sandhya et al., 2009). Nowadays, the improved PGPR strains had been introduced to
farmers to increase food production (ICAR Report, 2006-2007). These types of
findings are providing a new way of bio-farming that could be able to reduce the
burden of chemical fertilizers. Therefore, present study was aimed to find out the
PGPR that can play a crucial role as bio-fertilizer, bio-control and drought-tolerant
agents.
One way to identify and isolate drought-resistant PGPR is to find them from
plants that face drought, like Spilanthes acmella Murr., which is a threatened
medicinal plant. It is a well-known “toothache plant,” commonly known as Akarkara-
an annual herb, around 40 to 60 cm tall with prostrate stem in some of the species and
erect in others. It is found throughout India, ascending to 5000 ft., in all warm regions
(Tiwari et al., 2011) including Lower Shivalik Hills of Himachal Pradesh
(Singh and Thakur, 2014; Chauhan, 2006). It has a long history of folklore remedy for
toothache, rheumatism and fever (Wongsawatkul et al., 2008). The plant has found
applications in pharmaceuticals as an anti-toothache formulation for pain relief,
swelling, mouthwashes and gum infections (Pandey et al., 2007). Its extract is used in
cosmetics as a fast-acting muscle relaxant to accelerate the repair of wrinkles (Belfer,
2007). It is also used as a nutritional supplement for taste improvement as a sweetener
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Chapter 1 Introduction
with high sweetness devoid of unpleasant aftertaste that does not affect the taste or
odor of foods or drinks (Miyazawa et al., 2006). Several constituents had been
isolated from the S. acmella, for example, spilanthol, isobutylamides and
triterpenoids. S. acmella also exhibits vasorelaxant and antioxidant activities
(Wongsawatkul et al., 2008).
The roots, flower heads and whole aerial part of S. acmella Murr. yield a
powerful stimulant and local anesthetic compound known as spilanthol. In the
Ayurvedic system of medicine, flower heads and roots are used to treat scabies,
psoriasis, scurvy, toothache, gums and throat infections, paralysis of the tongue and
remedy for stammering in children (Nabi et al., 2016). It has been well documented
for its uses as an antibacterial, antifungal and antimalarial activity. Traditionally, the
plant is also used in the treatment of toothache, flu, cough and tuberculosis
(Yadav and Singh, 2010). Because of its high medicinal value, there is high demand
for this plant in the market.
Plant growth and productivity are adversely affected due to the abiotic stress
factor, i.e. drought. Plant growth-promoting rhizobacteria help to alleviate the effect
of drought stress by using direct and indirect mechanisms and ultimately enhances
plant growth and productivity. Keeping these facts in mind, the present study has been
undertaken to investigate the presence and potential of PGPR in the rhizospheric
region of S. acmella. Furthermore, analyzed their ability to act as PGP as well as for
alleviation of drought.

1.1 AIMS AND OBJECTIVES

1. To isolate the rhizospheric and endophytic bacterial strains from rhizosphere soil and
root samples of the wild population of Spilanthes acmella Murr.
2. To screen the bacterial strains that can survive under in vitro drought stress conditions
by analysis of growth in polyethylene glycol (PEG-6000).
3. To identify and characterize bacterial strains based on biochemical, 16S ribosomal
deoxyribonucleic acid (16S rDNA) sequencing and plant growth-promoting traits.
4. To analyse the effect of potential bacterial strains on alleviation of drought stress on
wheat seedlings under drought stress.

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