Study of Plant Growth Promoting Rhizobacteria in Earthworm Burrow Wall Soil
Transcript of Study of Plant Growth Promoting Rhizobacteria in Earthworm Burrow Wall Soil
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Study of Plant Growth Promoting Rhizobacteria in
earthworm burrow wall soil
Earthworms prepare the ground in an excellent manner for the growth of
fibrous rooted plants and for seedlings of all kinds.
Charles Darwin
INTRODUCTION
Plant growth-promoting rhizobacteria (PGPR) were first defined by Kloepper and
Schroth (1978) to describe soil bacteria that colonize the roots of plants following
inoculation onto seed and thereby enhance plant growth. Most PGPR are free-living
bacteria (Kloepper et al., 1989) and some invade the tissues of living plants causing
unapparent and asymptomatic infections (Sturz and Nowak 2000). These heterogenous
bacteria are associated with the rhizosphere, which is an important soil ecological
environment for plantmicrobe interactions. PGPR may induce plant growth promotion
by direct or indirect modes of action (Beauchamp 1993; Kloepper 1993; Kapulnik 1996;
Lazarovits and Nowak 1997). Direct mechanisms include the production of stimulatory
bacterial volatiles and phytohormones, lowering of the ethylene level in plant,
improvement of the plant nutrient status by liberating phosphates and micronutrients
from insoluble sources and stimulation of disease-resistance mechanisms. Indirect effects
are seen for example when PGPR act like biocontrol agents reducing diseases (Jacobsen,
1997). The beneficial effects of PGPRs have been attributed to biological N2 fixation
(Boddy et al., 1995; Meunchang et al., 2005) and production of phytohormones that
promote root development and proliferation resulting in more efficient uptake of water
and nutrients (Jacoud et al., 1999). These bacteria belong to the genera Azotobacter,Azospirillum, Bacillus, Arthrobacter, Enterobacter, Pseudomonas, Alcaligenes,
Klebsiella and Serratia (Dobereiner 1992).
Earthworms form a major component of the soil system and have been efficiently
ploughing the land for millions of years and assisting in the recycling of organic nutrients
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for the efficient growth of plants. Availability of earthworms in soils has always
promoted plant growth (Springet and Syers, 1979; Edwards and Lofty, 1980). Perhaps the
most well-known aspect of how earthworms affect plant growth is that they aerate the
soil as they tunnel through it to form burrows. Burrow diameters may range from less than
1 mm to greater than 10 mm, and extend as deep as 15 m and therefore, have profound
implications not only for aeration and water conductivity but also for channelling of plant
roots. Interactions between earthworms and microorganisms, in the degradation and
stabilization of organic wastes, to produce vermicomposts, can increase the potential
production of plant growth regulators, since this process increases microbial diversity,
populations and activity to a large extent. Scientists have reported the production of
grass, wheat and clover (Van Rhee 1965) to increase many fold by the presence ofearthworms, while increase in growth of maize, (Spain et al., 1992) paddy, sugarcane,
vegetables and ornamental plants (Kale et al., 1987) have also been reported. The
beneficial effect of earthworms on plant growth may be due to several reasons apart from
the presence of macronutrients and micronutrients in vermicasts and in their secretions in
considerable quantities. Plant growth promoting substances (e.g. vitamins, plant
hormones, enzymes and amino acids) have been detected in earthworm extracts (Graff
and Makeschin 1980; Dell'Agnola and Nardi 1987). In India, very early reports are
available on the chemical properties of earthworm castings that can play a positive role in
plant growth (Kale and Karmegam 2010).The combined effect of earthworms on (i) soil
structure, (ii) organic matter dynamics and (iii) nutrient release is, usually to stimulate
plant growth. Several studies have shown that this effect is positive (Derouard et al.,
1997; Gilot- Villenave et al., 1996; Stephens et al., 1994) even though not all plants
respond equally and the response is proportional to the earthworms biomass.
The mechanisms by which earthworms increase nutrient availability for plant growth are
still not very clear, but there are reports that most likely they depend on microbial
activities (Edwards and Lofty 1980; Parle 1963). Earthworms have been found to
stimulate soil enzymes, such as glucosidase and phosphatase (possibly of microbial
origin) which influence availability of plant nutrients (Ross and Cairns 1982; Tiwari et
al., 1989). Microbial derived plant hormones have also been isolated from earthworm
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casts (Tomati et al., 1988). Just like PGPRs are known to be activated by root exudates,
soil bacteria stimulated by earthworm mucus could act on plant physiology by emitting
phytohormones in the soil, which induces modifications of root system morphology
and/or systemic resistance to parasites. Nielson (1965) identified indole compounds in
extracts of several lumbricids while Springett and Syers (1979) suggested that auxin-like
substances are present in casts.
Indole-3-acetic acid, also known as IAA, is the member of the group of phytohormones
and is generally considered the most important native auxin (Ashrafuzzaman et al.,2009)
and is probably the most important plant auxin produced by PGPRs. It functions as an
important signal molecule in the regulation of plant development including
organogenesis, tropic responses, cellular responses such as cell expansion, division, and
differentiation, and gene regulation [Ryu and Patten 2008a). IAA has many different
effects with subsequent results for plant growth and development. The potential for auxin
biosynthesis by rhizobacteria can be used as a tool for the screening of effective PGPR
strains (Khalid et al., 2004). Even the strains, which produce low amounts of IAA,
release it continuously, thus improving plant growth [Tsavkelova et al.,2007). Some of
the IAA producing microorganisms include Acetobacter xylinum, Arthrobacter citreus,
Bacillus cereus, Pseudomonas aeruginosa, Xamthomonas maltophilia, Rhizobium
leguminosarum, Rhizobium japonicum andAzospirillum sp (Vessy, 2003).Ishmail (1995)
has reported plant hormone-like compounds (benzyladenine equivalents and IAA
equivalents) in casts ofL. maruitiiandP. excavates. Plant hormones derived from microbes
have also been isolated from earthworm casts (Tomati et al., 1988).
In the natural environment, PGPRs produce siderophores to acquire iron. Some PGPRs
can also utilize iron from heterologous siderophores produced by neighboring
microorganisms (Muragappan et al.,2006). Iron is required by aerobic bacteria and other
living organisms for a variety of biochemical reactions in the cell. Although iron is the
fourth most abundant element in the earth's crust, it is not readily available to bacteria.
Most of the aerobic organisms have developed an efficient means for solublizing and
transporting iron. The mechanism involves the role of low molecular weight organic
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compounds that have the ability to chelate iron and transport it across the cell. The many
different types of siderophores can generally be classified into two structural groups,
hydroxamates and catecholate compounds. Distribution of siderophore producing isolates
according to amplified ribosomal DNA restriction analysis (ARDRA) groups reveals that
most of the isolates belong to Gram- negative bacteria corresponding to thePseudomonas
and Enterobacter genera. Bacillus and Rhodococcus genera are the Gram-positive
bacteria found to produce siderophores (Tian et al., 2009).
Currently, there are many bacterial genera that include PGPR among them, revealing a
high diversity in this group. Some of the most abundant PGPR are as follows:
Diazotrophic PGPR - Free nitrogen-fixing bacteria were probably the first rhizobacteria
used to promote plant growth.Azospirillumstrains have been isolated and used since the
1970s (Steenhoudt and Vanderleyden 2000). Bashan et al., (2004) have reported the
latest advances in physiology, molecular characteristics and agricultural applications of
this genus. Other bacterial genera capable of nitrogen fixation that is probably
responsible for growth promotion effect, are Azoarcus spp., Burkholderia spp.,
Gluconacetobacter diazotrophicus, Herbaspirillum spp., Azotobacter spp. and Bacillus
polymyxa(Vessey 2003).
Denitrifying bacteria- They convert nitrate to nitrogen (N2) or nitrous oxide (N2O) gas.
Several species of Bacillus, for exampleB. pantothenticus,B. cereusandB. lactosporus
are capable of denitrification. Members of Bacillusspecies are able to form endospores
and hence survive under adverse conditions; some species are diazotrophs such as
Bacillus subtilis (Timmusk 1999), whereas others have different PGPR capacities, as
many reports on their growth promoting activity reveal (Kokalis-Burelle et al., 2002,
Probanza et al.,2001).Bacillus species have been reported to promote the growth of a
wide range of plants (De Freitas et al., 1997; Kokalis-Burelle et al., 2002); however, they
are very effective in the biological control of many plant diseases.
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Pseudomonas- among Gram-negative soil bacteria, Pseudomonas is the most abundant
genus and the PGPR activity of some of these strains has been known for many years,
resulting in a broad knowledge of the mechanisms involved (Lucas Garca et al., 2004;
Patten and Glick 2002). The ecological diversity of this genus is enormous, since
individual species have been isolated from a number of plant species in different soils
throughout the world. Pseudomonas strains show high versatility in their metabolic
capacity. Antibiotics, siderophores or hydrogen cyanide are among the metabolites
generally released by these strains (Charest et al., 2005). These metabolites strongly
affect the environment, both because they inhibit growth of other deleterious
microorganisms and because they increase nutrient availability for the plant. They secrete
pyoverdin (fluorescein), a fluorescent yellow-green siderophore (Meyer et al., 2002).
CertainPseudomonasspecies may also produce additional types of siderophore, such as
pyocyanin by P. aeruginosa (Lau et al., 2004) and thioquinolobactin by P. fluorescens
(Mattijs et al.,2007).
Rhizobia-Rhizobium well known for their beneficial symbiotic atmospheric nitrogen
fixing symbiosis with legumes has an excellent potential to be used as PGPR with non
legumes in a nonspecific relationship (Antoun et al., 1998). They form an endosymbiotic
nitrogen fixing association with roots of legumes. Here the bacteria converts atmospheric
nitrogen to ammonia and then provides organic nitrogenous compounds such as
glutamine or ureides to the plant (Sawada et al.,2003). It is well known that a number of
individual species may release plant growth regulators, siderophores and hydrogen
cyanide or may increase phosphate availability, thereby improving plant nutrition
(Antoun et al., 1998). Agrobacterium a free living rhizobacterium is closely related to
Rhizobium.
Ammonifying bacteria- These bacteria are significant for the biological process of
ammonifcation. Soil bacteria decompose organic nitrogen forms in soil to the ammonium
form. In soils NH3 is rapidly converted to NH4+
when hydrogen ions are plentiful.
Bacillus,Clostridium, Proteus and Pseudomonas are the bacteria which belong to this
group.
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Nitrifying bacteriaThese bacteria grow by consuming inorganic nitrogen compounds
and change ammonium (NH4+
) to nitrite (NO2-
) then to nitrate (NO3-
)a preferred form
of nitrogen for grasses and most row crops. Nitrifying bacteria belong to the genera
Nitrosomonas,Nitrosococcus,Nitrobacter andNitrococcus.
Enteric bacilli- Enteric bacteria are members of the family Enterobacteriaceae and
include:Eschericia,Enterobacter, Salmonella, Shigella,Proteusand Yersinia.
The present study aims at isolating and analyzing the PGPRs from the burrow wall soils
ofP. corethrurusandL. mauritii.
Materials and Methods
Generation of Soil Samples
The soil sample was generated as described in chapter 2.
Isolation of PGPRs on different media
PGPRs were isolated using the following media
Yeast Extract Mannitol Agar (YEMA) medium forRhizobiumandAgrobacterium Pseudomonas isolation agar (PIA) medium forPseudomonas Kleigler medium for Enteric bacilli Nitrogen free Malate medium forAzospirillum Nitrate medium for nitrifiers Ammonification medium for ammonifiers Ashbys medium forAzotobacter
1g soil samples were serially diluted and the dilution of 10-6
was plated on to the different
selective media. Kleigler and Pseuodomonas Isolation Agar (PIA) were observed after
24hrs of incubation whereas the rest of the media were observed after 2-3 days for the
growth of colonies.
The isolates were identified based on colony characteristics and Gram staining.
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Rhizobium and Agrobacterium on YEMA medium
Rhizobiumcolonies are round varying from flat to doomed and even conical shape having
smooth margins. In sub-surfaces they are typically lens shaped. They form white,
translucent, glistening, elevated and comparatively small colonies. Agrobacaterium
colonies are very similar to Rhizobium but have unique capability to take up congo red
when grown on YEMA. They appear as dark pink colonies. On Gram staining, both the
organisms appear as Gram negative rods.
Pseudomonas on PIA medium
The colonies appear as round with smooth margin, producing fluorescence of blue, green
and yellow. On Gram staining, they appear as Gram negative rods. They are oxidase
positive, motile, urease negative and give green fluorescence on Kings B medium.
Isolation of Enteric bacilli on Kleigler medium
This differential medium is commonly used to separate lactose fermenting members of
the family Enterobacteriaceae from members that do not ferment lactose, like Shigella.
The colonies appeared flat or slightly convex with irregular edges and ground-glass
appearance. On Gram staining, Gram negative rods are observed.
Isolation of Azotobacter on Ashbys media
The colonies on both media appear white, transparent and watery. They are round, doom
like with smooth surface and margin. On Gram staining, Gram negative rods can be
observed
Isolation of Ammonifying bacteriaWhite, brown colonies with slimy surface were observed. On Gram staining, both Gram
negative rods and Gram positive rods in chain with central spores are observed.
Isolation of denitrifiers of Nitrate medium
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The colonies are observed to be brown, white and translucent with rhizoid and smooth
margin. On Gram staining, organisms are observed to be both Gram negative rods and
Gram positive rods. Species ofNitrosomonasandNitrobacterare Gram negative, mostly
rod-shaped, microbes ranging between 0.6-4.0 microns in length.
Isolation of Azospirillum on Nitrogen free Malate medium
The growth of the organism can be confirmed by the colour change in the medium from
green to blue which is due to the change in the pH of the medium from acidic to neutral
to alkaline. White pellicle formation of 2-4 mm below the surface of the medium. Gram
negative vibriod bacteria can be observed on staining.
Quantitative analysis of IAA production by PGPRs
Indole acetic acid produced by bacteria was assayed colorimetrically using ferric chloride
perchloric acid reagent (FeCl3- HClO4) (Gordon and Weber 1951). This method
estimated the quantities of indole compounds produced by bacteria in the medium
containing precursor L- tryptophan.
Growth media- Luria- Bertani (LB) agar medium amended with 5Mm L- tryptophan
Reagents Orthophosphoric acid, FeCl3HClO41ml of 0.5M FeCl3 in 50 ml of 35%
HClO4, Stock: 100mg/ml of IAA in 50% ethanol.
A pink color develops when a mineral acid is added to a solution containing indole acetic
acid in the presence of ferric chloride. Different mineral acids, HCl, phosphoric acid,
nitric acid, sulfuric acid and perchloric acid can be used for development of color. FeCl3
HClO4 reagent is the most sensitive and shows least interference from other indole
compounds, example: tryptophan, skatol, acetyltryptamine. Since Beers law is not
followed at high concentrations of IAA, absorbences obtained are converted to IAA
concentration by a standard curve.
Procedure
Luria- Bertani (LB) broth medium amended with Tryptophane, was asepticallyinoculated with pure cultures of the isolates.
This was incubated at a 30oC for 24 hrs in rotary shaker (120 rpm).
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1.5 ml bacterial culture was centrifuged at 2,000 rpm for 5 minutes. To 1 ml of the supernatant 2ml of FeCl3- HClO4 reagent was added. After 25 minutes of incubation the absorbance was read in UV-
spectrophotometer at 530 nm.
The amount of IAA produced per milliliter culture was estimated using a standardcurve.
Study of Siderophore production
Growth mediaSuccinate Medium
Isolates were grown in the Succinate medium and incubated in a rotary incubatorat 37 C 150 rpm for 72 hours.
The culture was centrifuged at 2000 rpm for 5 minutes. The cell free supernatant was examined for absorption spectrum between 200-
600 nm using UV visible spectrophotometer.
The peak was determined by plotting the graph.
Results
The total count of various PGPRs isolated on specific media from P. corethrurusworked
soils is shown in Table 4.1. A significant decrease in the total count of Rhizobium and
Azotobacter was observed in the lower burrow wall soil of both 30 and 45 day trials
compared to control soil. There was no difference in the total count in the upper burrow
wall soils of both trails compared to the respective control soils. A similar result was also
observed in the case of total count of Agrobacteriumwhere a significant increase was
seen only in the 30 days upper burrow wall soil. The count of enteric bacilli showed a
significant increase in the 30 days lower burrow wall and 45 days upper burrow wall soil
compared to control. The number of Azospirillumsignificantly increased in both the 30
and 45 days lower burrow wall soil and significantly decreased in the 30 days upper
burrow wall soil.Pseudomonasshowed a significant decrease in both upper and lower 45
days samples and also in the 30 days lower burrow wall soil. In the 30 days upper burrow
wall there was a significant increase. Denitrifiers were observed to decrease significantly
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in all burrow wall soils except in the 30 days upper burrow wall soil. Ammonifiers
significantly increased in the 30 days burrow wall soil but decreased in the 45 days
borrow wall soil compared to control.
The total count of various PGPRs isolated on specific media from L. mauritii worked
soils is shown in Table 4.2. In L. mauritii worked soils it was observed that the total
count of Rhizobiumsignificantly increased in the 30 days trials in both upper and lower
burrow wall soil and the 45 days upper burrow wall soil. There was a decrease in the 45
days lower burrow wall soil. The total count of Agrobacterium also significantly
increased in the 45 days upper and lower burrow wall soil and 30 days lower burrow wall
soil. Enteric bacilli significantly increased in the 30 days upper and 45 days lower burrow
wall soil and decreased in the 30 days lower and 45 days upper burrow wall soil.
Azotobacter significantly increased in the 30 days lower and 45 days upper burrow wall
soil but decreased significantly in the 30 days upper and 45 days lower burrow wall soil.
Azospirillumwas not isolated from any of the soil samples in the study. Pseudomonas
significantly increased in the lower burrow wall soil of both 30 and 45 days trials
compared to control whereas in the upper burrow wall soil there was a significant
decrease. The total count of Pseudomonas was much higher than the other PGPRs
isolated. The total count of denitrifiers were significantly higher in the 30 days upper
burrow wall and 45 days upper and lower burrow wall soil compared to control. In the
lower burrow wall 30 days soil it significantly decreased. Ammonifiers showed a
significant increase in the 30 days burrow wall soil and a significant decrease in the 45
day upper burrow wall soil. There was no difference in the 45 days lower burrow wall
soil.
IAA production
The highest IAA production was seen in isolates from lower burrow wall soil (Graph
4.1). Strains ofAzotobacter (38.72mg/ml) andRhizobium(41.74mg/ml) isolated from 30
days lower burrow wall soil produced the maximum IAA followed by Pseudomonas
(34.43 mg/ml) isolated from 45 days upper burrow wall soil. Pseudomonasstrains from
30 days upper burrow wall (25.46 mg/ml) and 45 days lower burrow wall (34.43 mg/ml)
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soil also showed high levels of IAA production compared to isolates from control soils.
Rhizobium isolates from other samples did not show significant IAA production. The
enteric bacilli isolate from, 30 days lower burrow wall (24.99 mg/ml), upper burrow wall
(10.51 mg/ml) soil and denitrifiers (27.47mg/ml) also produced high amounts of IAA.
Siderophore production
All isolates fromP. corethrurusandL. mauritiishowed a peak between 240nm300nm
indicating the presence of catecholates type of siderophore.Pseudomonas,Rhizobiumand
enteric bacilli showed a peak at both 240nm and 450nm indicating the production of
mixed type of siderophore that is both hydroxamates (450 nm) and catecholates (240 nm)
type (Graph 4.2, 4.3, 4.4 and 4.5).
Discussion
PGPRs have gained worldwide importance and acceptance for agricultural benefits.
These microorganisms are the potential tools for sustainable agriculture and the trend for
the future.With new possibilities being opened up concerning the application of
beneficial bacteria to the soil for the promotion of plant growth and the biological control
of soil-borne pathogens and the large scale release of genetically engineered bacteria tothe environment facing a number of regulatory hurdles, the need to isolate and select
superior, naturally occurring PGPRs continue to be of interest. Making use of their
beneficial effects requires detailed knowledge on the diversity of PGPRs.
Our results show that the different earthworm species have different effect on the PGPRs.
The soil worked withP. corethrurusshowed a decrease in the total count of some PGPRs
in the burrow wall compared to their respective control whereas L. mauritii worked
burrow wall soils showed an increase in various PGPRs. From the burrow wall of P.
corethrurus and L. mauritii the 7 species of PGPRs were isolated viz.,Rhizobium,
Agrobacterium, Enteric Bacilli, Azotobacter, Pseudomonas, Denitrifiers and
Ammonifiers. Azospirillum was isolated only from the burrow wall ofP. corethrurus.In
a study by Bertrandet al., (2001), 13 Gram-negative bacteria were isolated from the
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rhizoplane and endorhizosphere ofBrassica napusand around ten bacterial PGPRs were
isolated from the rhizosphere soils of rice field from different areas of Mymensingh in
Bangladesh by Ashrafuzzaman et al., (2009). Studies by Akbari et al., (2007) showed
about 50 strains of Azospirillum isolated from plant roots of Iranian soil. There are no
earlier reports of studies on PGPRs from earthworm burrow walls.
InP.corethrurusburrow wall soils an overall increase in Azospirillumand enteric bacilli
was observed in this study whereas decrease inRhizobiumandAgrobacteriumwas seen.
In L. mauritii burrow wall soils both Rhizobium and Agrobacterium increased in
number.The lower and upper burrow wall soils showed difference in the total count of
Pseudomonaswhich significantly increased in the lower burrow wall and decreased in
the upper burrow wall soil. This indicates that depth too has an impact on the association
of earthworms and distribution of PGPRs. OverallRhizobiumand denitrifiers increased in
the 30 days burrow wall soil. Agrobacterium and denitrifiers increased in the 45 days
burrow wall soil.
The PGPRs isolated from the burrow wall ofL. mauritiiproduced IAA ranging from 4.42
mg/ml 41.74mg/ml the highest being in strains of Azotobacter and Rhizobium.
Meunchang et al., (2006), have reported the IAA production ranging from 10-69mg/ml
by Rhizobacteria and around 29mg/ml by indigenous Azospirillum spp isolated from
Irannian soils by Akbari et al., (2007). Rhizobia are the first group of bacteria, which are
attributed to the ability of PGPR to release IAA that can help to promote the growth and
pathogenesis in plants [Mandal et al.,2007.) Sridevi and Mallaiah (2007) showed that all
the strains of Rhizobium isolated from root nodules of Sesbania sesban (L) Merr.
produces IAA. Reports also show that all strains of Bacillus, Pseudomonas and
Azotobacter associated with chickpeaproduced IAA, whereas only 85.7% of Rhizobium
was able to produce IAA (Joseph et al.,2007). Isolates producing IAA have stimulatory
effect on the plant growth and the fact that strains from burrow wall soil produced high
IAA is significant. In a study by Ahmad et al.,(2005) showed 5 isolates ofPseudomonas
producing high levels (41.0 to 53.2 mg/ml) of IAA while 6 other isolates produced IAA
in the range of 23.4 to 36.2 mg/ml (Ahmad et al.,2005). Production of high levels of IAA
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by fluorescent Pseudomonas is a general characteristic; the burrow wall isolates showed a
similar high level of IAA production to those recorded by other researchers (Caron et al.,
1995; Frankenberger and Poth 1989; Glick 1995).
The interactions between earthworms and microorganisms can produce significant
quantities of plant growth hormones and plant regulators. The wide variety of
siderophores may be due to evolutionary pressures placed on microbes to produce
structurally different siderophores which cannot be transported by other microbes'
specific active transport systems, or in the case of pathogens deactivated by the host
organism. Siderophores can also suppress plant diseases by reducing the availability of
Fe deleterious microbes and their role in plant growth and biological control is well
established (Hass and Defago, 2005). Mayer and Abdullah (1978) have reported mixed
type of siderophores (hydroxamates and catecholate) produced by Pseudomonas. The
present study also showed a mixed type of siderophore production by Pseudomonas
isolates from both P. corethrurus and L. mauritii. Marianne and Page (1988) have
reported the production of the catechol siderophores when Azotobacter vinelandiiwas
grown in the presence of low levels of iron. Azotobacter from this study also showed
catechol type of siderophores. Rhizobium strains isolated from the root nodules of the
Sesbania sesban (L) Merr. show the ability to produce hydroxamate-type of siderophores
(Sridevi and Mallaiah 2008). Rhizobial isolates belonging to generaRhizobium sp. and
Mesorhizobium sp. produces only catecholate type of siderophores (Joshi et al.,2009).
Most strains ofRhizobiumandAzotobacterfrom this study also showed only catecholate
type of siderophores.
Through their numerous direct or indirect mechanisms of action, PGPR can allow
significant reduction in the use of pesticides and chemical fertilizers. These beneficial
events producing biological control of diseases and pests, plant growth promotion,
increases in crops yield and quality improvement, can take place simultaneously or
sequentially. The presence of earthworms in the soil is often considered to be a positive
indicator of soil quality and productivity. The relationship between plant roots and
earthworm burrows is complex, with some plant roots preferentially exploring earthworm
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burrows emphasizing that earthworm burrow walls could be a significant source of
PGPRs. The variability in the performance of PGPR may be due to various
environmental factors that may affect their growth and exert their effects on plant. The
environmental factors include climate, weather conditions, soil characteristics or the
composition or activity of the indigenous microbial flora of the soil. Therefore, it is
necessary to develop efficient strains in field conditions. One possible approach is to
explore soil microbial diversity for PGPR having combination of PGP activities and well
adapted to particular soil environment and the present study is a step in this direction.
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Legend: LBWS- Lower Burrow Wall Soil; LCS- Lower Control Soil; UBWS- Upper Burrow Wall
Soil; UCS- Upper Control Soil
Figure 4.2: Absorption maxima of siderophores from isolates of Pseudomonas,
Enteric bacilli andAzotobacterfrom burrow wall soil of L. mauritii
Legend: LBWS- Lower Burrow Wall Soil; LCS- Lower Control Soil; UBWS- Upper Burrow Wall
Soil; UCS- Upper Control Soil
Figure 4.3: Absorption maxima of siderophores from isolates of Denitrifiers and
Rhizobiumfrom burrow wall soil of L. mauritii
0
0.5
1
1.5
2
2.5
3
3.5
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
OpticalDensity
Wave length nm
UBWS 30 d (Pseudomonas)
UBWS 45 d (Pseudomonas)
LBWS 45 d (Pseudomonas)
UBWS 30 d (Enteric bacilii)
UBWS 30 d (Azotobacter)LBWS 30 d (Azotobacter)
UBWS 45 d (Azotobacter)
LBWS 45 d (Azotobacter)
0
0.5
1
1.5
2
2.5
3
3.5
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
OpticalDensity
Wave length nm
UBWS 30d (Denitrifiers)
LBWS 30d (Denitrifiers)
UBWS 45d (Denitrifiers)
LBWS 45 d (Denitrifiers)
UBWS 30 d (Rhizobium)
LBWS 30 d (Rhizobium)
LBWS 45 d (Rhizobium)
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Legend: LBWS- Lower Burrow Wall Soil; LCS- Lower Control Soil; UBWS- Upper Burrow Wall
Soil; UCS- Upper Control Soil
Figure 4.4: Absorption maxima of siderophores from isolates of Rhizobium,
Azotobacterand Denitrifiers from burrow wall soil of P. corethrurus
Legend: LBWS- Lower Burrow Wall Soil; LCS- Lower Control Soil; UBWS- Upper Burrow Wall
Soil; UCS- Upper Control Soil
Figure 4.5: Absorption maxima of siderophores from isolates of Ammonifiers,
Enteric bacilli and Pseudomonasfrom burrow wall soil of P.
corethrurus
0
0.5
1
1.5
2
2.5
3
3.5
4
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
OpticalDensity
Wave length nm
LBWS 45 d (Azotobacter)
LBWS30d (Rhizobium)
UBWS 30 d(Rhizobium)
LBWS 45 d(Rhizobium)
UBWS 45d (Denitrifiers)LBWS 45d (Denitrifiers)
LBWS 30d (Denitrifiers)
0
0.5
1
1.5
2
2.5
3
3.5
4
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600
OpticalDensity
Wave length nm
LBWS 30 d (Ammonifiers)
UBWS 45 d (Ammonifiers)
UBWS 30 d (Ammonifiers)
LBWS 45d (Enteric bacilli)
LBWS 30d (Enteric bacilli)
UCS 45d (Enteric bacilli)
UBWS 30 D(Pseudomonas)
LBWS 30 d (Pseudomonas)
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Table 4.1: PGPRs isolated on different media from burrow wall soil of P. corethrurusand control soil
Rhizobium(x10 7)
Agrobacterium(x10 7)
Enteric Bacilli(x10 7)
Azospirillum(x10 7)
Azotobacter(x10 7)
Pseudomonas(x10 6)
Denitrifiers(x10 7)
Ammonifiers(x106)
LBWS
(30 days)0.05c 0.04 0.58 de 0.55 4.15e 0.06 2.61a 0.05 0.64 b 0.002 0.4 e 0.02 3.1c 0.48 12.4 b 0.84
LCS
(30 days) 7.3 b 0.43 25.5 b 0.75 2.8f 0.02 2.22 b 0.09 1.7 a 0.11 4.09 d 0.35 14.16a 0.37 0.31 cd 0.08
UBWS
(30 days)0.5 c 0.23 9.45 c 0.43 0.25g 0.003 0.30 d 003 0.58 bc 0.27 6 c 0.26 1.03ef 0.13 0.67 cd 0.12
UCS
(30 days)0c 0.05e 0.04 0.11 g 0.09 0.48 c 0.01 0.33 cd 0.33 4 d 0.56 0.56fg 0.06 0.08 d 0.36
LBWS
(45 days)0.12c 0.06 0.11e 0.01 31.07d 0.62 2.2 b 0.05 0.09 de 0.08 0.6e 0.30 2.33 d 0.11 0.88 c 0.30
LCS
(45 days)10.0a 0.91 34.2 a 0.42 37.21c 0.10 0.09 e 0.01 1.81a 0.02 34a 1.55 11.46 b 0.51 12.22 b 0.38
UBWS
(45 days)0.16 c 0.04 0.06 e 0.001 50.6 a 0.3 0.06 e 0.02 0.152 de 0.05 4.5d 0.48 0.08 g 0.04 0.33 cd 0.07
UCS
(45 days)0.70 c 0.14 0.91 d 0.03 44.12 b 0.12 0.09 e 0.01 0.02 e 0.009 10.3b 0.56 1.12 e 0.06 16.1 a 0.44
Means with same superscript in each column do not differ significantly at P
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Table 4.2: PGPRs isolated on different media from burrow wall soil of L. mauritiiand control soil
Rhizobium
(x 107)
Agrobacterium
(x 107)
Enteric Bacilli
(x 107)
Azospirillum
(x 107)
Azotobacter
(x 107)
Psuedomonas
(x 109)
Denitrifiers
(x 107)
Ammonifiers
(x 107)
LBWS
(30 days)
6.45 b 0.59 1.6 b 0.46 5.4 d 0.92 Nil 4.46 d 0.82 0.07 c 0.04 18.78 b 0.62 7.46 a 0.88
LCS
(30 days)2.86 d 0.48 1.08 bc 0.12 7.3 c 0.55 Nil 2.46 e 0.69 2.15 a 0.55 95.29 a 0.30 4.2 de 0.76
UBWS
(30 days)9.86 a 0.55 1.26 bc 0.52 11.7 a 0.84 Nil 9.46 b 0.84 2.06 a 0.72 9.49 d 0.84 8.13 a 0.95
UCS
(30 days)5.93 b 0.81 1.4 bc 0.40 3.13 e 0.33 Nil 12.22 a 0.5 1.34 b 0.52 4.12 f 0.52 5.86 bc 0.80
LBWS
(45 days)2.86 d 0.55 1.6 b 0.44 9.66 b 0.5 Nil 3.5 d 0.92 1.03 b 0.30 18.1 b 0.17 3.33 e 0.69
LCS
(45 days)3.5 c 0.46 0.86 c 0.36 2.52 e 0.47 Nil 10.2 b 0.79 3.91 a 0.25 5.39 e 0.56 3.33 e 0.61
UBWS
(45 days)6.13 b 0.73 3.6 a 0.03 2.93 e 0.28 Nil 10.1 b 0.91 3.66 a 0.65 12.2 c 0.78 4.9 cd 0.66
UCS
(45 days)2.41 d 0.56 1.13 bc 0.15 5.6 d 0.57 Nil 7.6 c 0.81 1.3 b 0.39 5.13 e 0.65 6.8 ab 0.63
Means with same superscript in each column do not differ significantly at P
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Plate 8