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

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 of earthworms, 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

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 and Azospirillum sp (Vessy, 2003). Ishmail (1995) has reported plant hormone-like compounds (benzyladenine equivalents and IAA equivalents) in casts of L. maruitii and P. 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

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 the Pseudomonas 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. Azospirillum strains 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 example B. pantothenticus, B. cereus and B. lactosporus are capable of denitrification. Members of Bacillus species 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.

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). Certain Pseudomonas species 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.

Nitrifying bacteria These 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 and Nitrococcus.

Enteric bacilli- Enteric bacteria are members of the family Enterobacteriaceae and include: Eschericia, Enterobacter, Salmonella, Shigella, Proteus and Yersinia.

The present study aims at isolating and analyzing the PGPRs from the burrow wall soils of P. corethrurus and L. 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 for Rhizobium and Agrobacterium Pseudomonas isolation agar (PIA) medium for Pseudomonas Kleigler medium for Enteric bacilli Nitrogen free Malate medium for Azospirillum Nitrate medium for nitrifiers Ammonification medium for ammonifiers Ashbys medium for Azotobacter

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.

Rhizobium and Agrobacterium on YEMA medium Rhizobium colonies 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 bacteria White, 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

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 of Nitrosomonas and Nitrobacter are 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, FeCl3HClO4 1ml 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 aseptically inoculated with pure cultures of the isolates. This was incubated at a 30oC for 24 hrs in rotary shaker (120 rpm).

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 standard curve.

Study of Siderophore production Growth media Succinate Medium Isolates were grown in the Succinate medium and incubated in a rotary incubator at 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 200600 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. corethrurus worked 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 Agrobacterium where 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 Azospirillum significantly increased in both the 30 and 45 days lower burrow wall soil and significantly decreased in the 30 days upper burrow wall soil. Pseudomonas showed 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

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 Rhizobium significantly 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. Azospirillum was 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 of Azotobacter (38.72mg/ml) and Rhizobium (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. Pseudomonas strains from 30 days upper burrow wall (25.46 mg/ml) and 45 days lower burrow wall (34.43 mg/ml)

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 from P. corethrurus and L. mauritii showed a peak between 240nm 300nm indicating the presence of catecholates type of siderophore. Pseudomonas, Rhizobium and 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 to the 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 with P. corethrurus showed 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 of P. corethrurus. In a study by Bertrand et al., (2001), 13 Gram-negative bacteria were isolated from the

rhizoplane and endorhizosphere of Brassica napus and 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.

In P.corethrurus burrow wall soils an overall increase in Azospirillum and enteric bacilli was observed in this study whereas decrease in Rhizobium and Agrobacterium was 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 Pseudomonas which 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. Overall Rhizobium and 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 of L. mauritii produced 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 chickpea produced 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 of Pseudomonas 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

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 vinelandii was 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 genera Rhizobium sp. and Mesorhizobium sp. produces only catecholate type of siderophores (Joshi et al., 2009). Most strains of Rhizobium and Azotobacter from 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

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.

45 40 35 30 IAA mg/ml 25 20 Pseudomonas Azotobacter Rhizobium Enteric Bacilli Denitrifiers

15
10 5 0
LBWS 30 LCS 30 UBWS 30 UCS 30 LBWS 45 LCS 45 UBWS 45 UCS 45

Samples

Legend: LBWS- Lower Burrow Wall Soil; LCS- Lower Control Soil; UBWS- Upper Burrow Wall Soil; UCS- Upper Control Soil

Figure 4.1: IAA production by isolates from burrow wall soil of L. mauritii and P. corethrurus.

3.5 3 2.5

Optical Density

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)

1.5 1 0.5 0 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

Wave length nm

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 and Azotobacter from burrow wall soil of L. mauritii

3.5 3 2.5
Optical Density

1.5 1 0.5 0

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)

200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

Wave length nm

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 Rhizobium from burrow wall soil of L. mauritii

4 3.5 3 Optical Density 2.5 2

LBWS 45 d (Azotobacter) LBWS30d (Rhizobium) UBWS 30 d(Rhizobium) LBWS 45 d(Rhizobium) UBWS 45d (Denitrifiers) LBWS 45d (Denitrifiers) LBWS 30d (Denitrifiers)

1.5 1

0.5 0
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

Wave length nm

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, Azotobacter and Denitrifiers from burrow wall soil of P. corethrurus

4 3.5 3
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)

Optical Density

2.5 2

1.5 1 0.5 0
200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600

Wave length nm

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 Pseudomonas from burrow wall soil of P. corethrurus

Table 4.1: PGPRs isolated on different media from burrow wall soil of P. corethrurus and control soil
Rhizobium (x10 7) LBWS (30 days) LCS (30 days) UBWS (30 days) UCS (30 days) LBWS (45 days) LCS (45 days) UBWS (45 days) UCS (45 days)
0.05c 0.04 7.3 b 0.43 0.5 c 0.23 0c 0.12c 0.06 10.0a 0.91 0.16 c 0.04 0.70 c 0.14

Agrobacterium (x10 7)
0.58 de 0.55 25.5 b 0.75 9.45 c 0.43 0.05e 0.04 0.11e 0.01 34.2 a 0.42 0.06 e 0.001 0.91 d 0.03

Enteric Bacilli (x10 7)

4.15e 0.06 2.8f 0.02 0.25g 0.003 0.11 g 0.09 31.07d 0.62 37.21c 0.10 50.6 a 0.3 44.12 b 0.12

Azospirillum (x10 7)
2.61a 0.05 2.22 b 0.09 0.30 d 003 0.48 c 0.01 2.2 b 0.05 0.09 e 0.01 0.06 e 0.02 0.09 e 0.01

Azotobacter (x10 7)
0.64 b 0.002 1.7 a 0.11 0.58 bc 0.27 0.33 cd 0.33 0.09 de 0.08 1.81a 0.02 0.152 de 0.05 0.02 e 0.009

Pseudomonas (x10 6)
0.4 e 0.02 4.09 d 0.35 6 c 0.26 4 d 0.56 0.6e 0.30 34a 1.55 4.5d 0.48 10.3b 0.56

Denitrifiers (x10 7)
3.1c 0.48 14.16a 0.37 1.03ef 0.13 0.56fg 0.06 2.33 d 0.11 11.46 b 0.51 0.08 g 0.04 1.12 e 0.06

Ammonifiers (x106)
12.4 b 0.84 0.31 cd 0.08 0.67 cd 0.12 0.08 d 0.36 0.88 c 0.30 12.22 b 0.38 0.33 cd 0.07 16.1 a 0.44

Means with same superscript in each column do not differ significantly at P<0.05 level by Duncans Multiple Range Test (DMRT). Legend: LBWS- Lower Burrow Wall Soil; LCS- Lower Control Soil; UBWS- Upper Burrow Wall Soil; UCS- Upper Control Soil

Table 4.2: PGPRs isolated on different media from burrow wall soil of L. mauritii and control soil
Rhizobium (x 10 7) LBWS (30 days) LCS (30 days) UBWS (30 days) UCS (30 days) LBWS (45 days) LCS (45 days) UBWS (45 days) UCS (45 days)
6.45 b 0.59 2.86 d 0.48 9.86 a 0.55 5.93 b 0.81 2.86 d 0.55 3.5 c 0.46 6.13 b 0.73 2.41 d 0.56

Agrobacterium (x 10 7)
1.6 b 0.46 1.08 bc 0.12 1.26 bc 0.52 1.4 bc 0.40 1.6 b 0.44 0.86 c 0.36 3.6 a 0.03 1.13 bc 0.15

Enteric Bacilli (x 10 7)
5.4 d 0.92 7.3 c 0.55 11.7 a 0.84 3.13 e 0.33 9.66 b 0.5 2.52 e 0.47 2.93 e 0.28 5.6 d 0.57

Azospirillum (x 10 7)
Nil Nil Nil Nil Nil Nil Nil Nil

Azotobacter (x 10 7)
4.46 d 0.82 2.46 e 0.69 9.46 b 0.84 12.22 a 0.5 3.5 d 0.92 10.2 b 0.79 10.1 b 0.91 7.6 c 0.81

Psuedomonas (x 10 9)
0.07 c 0.04 2.15 a 0.55 2.06 a 0.72 1.34 b 0.52 1.03 b 0.30 3.91 a 0.25 3.66 a 0.65 1.3 b 0.39

Denitrifiers (x 10 7)
18.78 b 0.62 95.29 a 0.30 9.49 d 0.84 4.12 f 0.52 18.1 b 0.17 5.39 e 0.56 12.2 c 0.78 5.13 e 0.65

Ammonifiers (x 10 7)
7.46 a 0.88 4.2 de 0.76 8.13 a 0.95 5.86 bc 0.80 3.33 e 0.69 3.33 e 0.61 4.9 cd 0.66 6.8 ab 0.63

Means with same superscript in each column do not differ significantly at P<0.05 level by Duncans Multiple Range Test (DMRT). Legend: LBWS- Lower Burrow Wall Soil; LCS- Lower Control Soil; UBWS- Upper Burrow Wall Soil; UCS- Upper Control Soil

Plate 8