study of plant growth promoting rhizobacteria in earthworm burrow wall soil

8/13/2019 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



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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Figure 4.4: Absorption maxima of siderophores from isolates of Rhizobium,

Azotobacterand Denitrifiers from burrow wall soil of P. corethrurus



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


21/21

Plate 8

study of plant growth promoting rhizobacteria in earthworm burrow wall soil

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