Assessing the Effect of Siderophore Producing Bacteria for the Iron Nutrition in Lentil And

ASSESSING THE EFFECT OF SIDEROPHORE PRODUCING

BACTERIA FOR THE IRON NUTRITION IN LENTIL AND PEA

A Thesis Submitted to the College of Graduate and Postdoctoral Studies in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Department of Soil Science University of Saskatchewan Saskatoon, SK, Canada

By Md Mortuza Reza

PERMISSION TO USE

In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department of

Soil Science or the Dean of the College of Agriculture and Bioresources. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use that may be made of any material in my thesis. Requests for permission to copy or to make other uses of materials in this thesis, in whole or part, should be addressed to:

Head, Department of Soil Science University of Saskatchewan Saskatoon, Saskatchewan Canada, S7N 5A8 Or

Dean, College of Graduate Studies and Research University of Saskatchewan Saskatoon, Saskatchewan Canada S7N 5A2

DISCLAIMER

Reference in this dissertation to any specific commercial products, process, or service by trade name, trademark, manufacturer, or otherwise, does not constitute or imply its endorsement, recommendation, or favouring by the University of Saskatchewan. The views and opinions of the author expressed herein do not state or reflect those of the University of Saskatchewan, and shall not be used for advertising or product endorsement purposes.

ABSTRACT

Lentil (Lens culinaris Medik., cv. CDC Milestone) was grown for 45 days in a growth

chamber using eight Saskatchewan agricultural soils to isolate root associated siderophore- producing and total heterotrophic bacteria. Results indicated that siderophore-producing bacteria and total heterotroph populations ranged between 4.17–6.30 log cfu·g-1 and 7.16–8.08 log cfu·g-

1, respectively. The in vitro siderophore production assay of 491 siderophore-producing isolates

using SideroTec® kit revealed that siderophore production ranged from 0.04 to 119.27 µg∙mL-1.

Isolates that exhibited high siderophore producing potential (n=68) were characterized using

Sanger sequencing and were identified as: Bacillaceae, Burkholderiaceae, Enterobacteriaceae,

Oxalobacteraceae, Pseudomonadaceae, Rhizobiaceae and Xanthomonadaceae. Next, isolates

that exhibited higher siderophore production (n=40) were inoculated on lentil to enhance plant

Fe uptake when grown in a sand-soil mixture. Results revealed that 33 isolates significantly increased total Fe uptake in lentil plants, but the ability to increase total Fe uptake in lentil was not associated with the ability of in vitro siderophore production of individual isolates. The most

effective five isolates of this study were subsequently tested to enhance Fe uptake in four pea cultivars in a growth chamber study. Results indicated that inoculation significantly increased total Fe uptake in 75% treatments. A study involving phytosiderophore production by four lentil

and four pea cultivars was conducted to assess the potential to chelate Fe from nutrient medium

during germination. Results of this study, indicated that all lentil and one pea, cultivar CDC

Dakota, exhibited positive phytosiderophore activity. However, results from the pea growth

chamber study indicated that the pea cultivar that exhibited in vitro phytosiderophore production

did not significantly enhance Fe uptake when compared to the remaining non phytosiderophore-

iii producing cultivars. This difference might be attributed to the differences between plant growth conditions and/or plant physiological stages.

Overall, significant increases in total Fe uptake by lentil and pea inoculated with siderophore-producing bacteria were observed. Moreover, the significant interactions between bacterial isolates and pea cultivars suggesting that plant species and cultivars might be dependent on their associated siderophore-producing isolates or vice versa for Fe nutrition and plant cultivars were more responsive when inoculated with their suitable siderophore-producing isolates.

ACKNOWLEDGEMENTS

To begin with, I would like to acknowledge my co-supervisors, Dr. Fran Walley and Dr. Renato de Freitas for every support and guidance they have given throughout this study. The incredible level of effort and enthusiasm you put into a student are highly appreciated.

I would like to express sincere gratitude to my advisory committee members, Professors, Drs. Jim Germida and Albert Vandenberg for their valuable advice, useful suggestions and feedbacks throughout my school and research project. I would thank Dr. Russell Hynes for reviewing my thesis as an external examiner and for his time and comments to improve the quality of this thesis.

This thesis work would not have been possible without the help of the members of Soil Agronomy (5C29) and Soil Microbiology (5E25) Labs. Special thanks to Jorge Cordero for his support in Microbiology Lab is media preparation and isolation. I am very grateful to Dr. Jeff Schoenau for providing me soils from his farm. Special thanks to Brent Barlow and Thiago Prado at the Crop Development Centre for providing me soils and quality seeds for my growth chamber studies. I would also like to thank Cory Fatteicher to Ranjan Kar for guidance regarding plant digestion processing for analysis. Thanks to all the friends and personnel at College of Agriculture and Bioresources for making the days in the university so pleasant.

I couldn’t have done this without the inspiration and supports of my family and friends, I am very thankful to all of you who provided much needed support in completing work. Thanks to my parents, Nural Islam and Morsheda Begum for encouraging and supporting me in all my undertakings. Thanks to the University of Saskatchewan and the Government of Saskatchewan for their financial support to carryout this research project.

DEDICATION

I would also like to dedicate this work to my father Nural Islam for his amazing encouragement. Finally, to my supervisors Dr. Fran Walley, Dr. Renato de Freitas and all the teachers around the world who help students to become successful.

TABLE OF CONTENTS

PERMISSION TO USE ...... i DISCLAIMER ...... ii ABSTRACT ...... iii ACKNOWLEDGEMENTS ...... v TABLE OF CONTENTS ...... vii LIST OF TABLES ...... x LIST OF FIGURES ...... xi LIST OF ABBREVIATIONS ...... xii 1. GENERAL INTRODUCTION ...... 1 2. LITERATURE REVIEW ...... 5 2.1 Importance of iron in human animal and plant ...... 5 2.2 Availability of Fe for the plants in soil ...... 7 2.3 Mechanisms of Fe-chelation by crop associated bacteria in soil ...... 8 2.4 Siderophores ...... 9 2.5 Effect of siderophore producing bacteria on Fe-nutrition and growth promotion of crop ...... 10 2.6 Isolation of siderophore-producing microorganisms ...... 12 2.7 Methods to measure siderophore production ...... 12 2.8 Methods for identification of the microbes ...... 14 3. ISOLATION AND IDENTIFICATION OF SIDEROPHORE PRODUCING BACTERIA FROM RHIZOSPHERE AND ENDORHIZOSPHERE OF LENTIL PLANTS GROWN IN DIFFERENT SASKATCHEWAN SOILS ...... 17 3.1 Preface ...... 17 3.2 Abstract ...... 17 3.3 Introduction ...... 18

3.4 Materials and methods ...... 20

3.4.1 Soil collection and growth chamber study ...... 20

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3.4.2 Isolation of siderophore-producing and total heterotrophic bacteria from rhizosphere and endorhizosphere using culture dependent method...... 21 3.4.3 Measuring siderophore production capacity of isolated root associated bacteria in vitro ...... 25 3.4.4 Identification of siderophore producing bacteria ...... 26 3.4.5 Statistical analysis ...... 26 3.5 Results ...... 26 3.5.1 Abundance of culturable siderophore producing bacteria ...... 26 3.5.2 Siderophore production by individual isolates ...... 30 3.5.3 Identification of siderophore producing bacteria ...... 32 3.6 Discussion ...... 35 3.7 Summary ...... 38 4. Effect of siderophore producing bacteria on iron content in lentil (Lens Culinaris MEDIK, cv. CDC Milestone) under controlled environmental conditions ...... 40 4.1 Preface ...... 40 4.2 Abstract ...... 40 4.3 Introduction ...... 41 4.4 Materials and methods ...... 44 4.4.1 Soil sampling and properties ...... 44 4.4.2 Measuring bacterial siderophore production ...... 45 4.4.3 Seed inoculation and plant growth ...... 45 4.4.4 Statistical analysis ...... 46 4.5 Results ...... 47 4.6 Discussion ...... 53 4.7 Summary ...... 55 5. EFFECT OF SIDEROPHORE-PRODUCING BACTERIA ON IRON CONTENT IN LENTIL AND PEA CULTIVARS ...... 57 5.1 Preface ...... 57 5.2 Abstract ...... 57 5.3 Introduction ...... 58

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5.4 Materials and methods ...... 62 5.4.1 Soil preparation and seed collection ...... 62 5.4.2 Phytosiderophore production assay ...... 63 5.4.3 Growth chamber assessment of growth enhancement by siderophore -producing bacteria ...... 63 5.4.4 Statistical analysis ...... 64 5.5 Results ...... 65 5.5.1 Phytosiderophore production assay ...... 65 5.5.2 Growth chamber assessment of growth enhancement by siderophore -producing bacteria ...... 67 5.6 Discussion ...... 71 5.7 Summary ...... 76 6. SYNTHESIS AND CONCLUSIONS ...... 77 6.1 Future perspectives ...... 80 7. REFERENCES ...... 81 8. APPENDIX ...... 100

LIST OF TABLES

Table 3.1 Physico-chemical properties of soil samples collected from different locations in Saskatchewan in 2013. All analyses were analyzed at ALS Environmental Laboratory, Saskatoon ...... 22 Table 3.2 Ranking of siderophore-producing isolates and identification according to Sanger (16S rRNA gene) sequencing ...... 33 Table 4.1 Final screening of selected (n=40) siderophore producing candidates for siderophore production (µg·mL-1) ± SD in vitro ...... 49 Table 4.2 Effect of siderophore producing bacteria inoculation on Fe uptake in lentil cv. CDC Milestone. Adapted from table 4.1 ...... 51 Table 5.1 Iron uptake in pea plants inoculated with siderophore-producing bacteria, grown in Brown (Ardill association) soil for 45 DAP in a growth chamber ...... 68 Table 5.2 Effect of siderophore producing bacteria inoculation on total iron uptake in different pea cultivars ...... 69

LIST OF FIGURES Fig. 3.1 Isolation of siderophore producing bacteria on Chrome Azurol S (CAS) medium. Orange halo represents the production of siderophore ...... 24 Fig. 3.2 Abundance of culturable siderophore-producing bacteria (colony forming unit = cfu) in selected Saskatchewan soils assessed on Chrome Azurol S medium. Different letters indicate significant differences using Tukey’s post hoc test at α=0.05 ...... 27 Fig. 3.3 Abundance of culturable total heterotrophic bacteria (colony forming unit = cfu) in selected Saskatchewan soils assessed on Trypticase soy agar (TSA) medium. Different letters indicate significant differences using Tukey’s post hoc test at α=0.05 ...... 28 Fig. 3.4 Percentage of siderophore-producing bacteria in selected Saskatchewan soils in relation to total heterotrophs ...... 29 Fig. 3.5 Measuring siderophore concentration using SideroTec Assay kit. Standard solution came with the kit ...... 30

Fig. 3.6 Box plots showing the siderophore concentration (µg·mL-1) for each location produced by isolates in Fe free M9 medium supernatants. Incubation was carried out at 28°C for 72h. The line in the box represents the median, the top and bottom of the box represent the 75th and 25th percentile of the data, respectively. The upper quartile and the lower quartile range were 1.5 times the interquartile range. Values more than 1.5 times the interquartile range are presented as dots (●), rhizosphere and endorhizosphere isolate numbers (n) were included for each location ...... 31 Fig. 5.1a Lentil cultivars CDC Dazil, CDC Milestone, CDC 3646-4 and CDC Maxim (A) and pea cultivars CDC Striker, CDC Dakota, CDC Meadow and CDC Amarillo (B) on Chrome Azurol S medium during germination at 5 days ...... 66 Fig. 5.1b In vitro siderophore production by isolates Li3E20 and Ca2E4 on Chrome Azurol S medium ...... 66 Fig. 5.2 Principal component analysis (PCA) of nutrient concentration and uptake patterns in 45 DAP pea cultivars CDC Amarillo, CDC Dakota, CDC Meadow and CDC Striker inoculated with 5 siderophore-producing bacteria isolates. On the x axis is the percentage of variation explained by principal component 1 (57.5%); on the y axis is the percentage of variation explained by principal component 2 (30.9%) ...... 70

LIST OF ABBREVIATIONS

ACC 1-aminocyclopropane-1-carboxylic acid BLAST Basic local alignment search tool CAS Chrome Azurol S cfu Colony forming units DAP Days after planting DGGE Denaturing gradient gel electrophoresis EDDHA Ethylenediamine di o-hydroxyphenylacetic acid EDTA Ethylenediamine tetraacetic acid FAMEs Fatty acid methyl esters FC Field capacity HDTMA Hexadecyl trimethyl ammonium bromide HPLC-MS High performance liquid chromatography-Mass spectrometry Fe Iron NCBI National center for biotechnology information OM Organic matter PBS Phosphate buffered saline PLFA Phospholipid fatty acid PIPES Piperazine-N,N'-bis[2- ethanesulfonic acid] PGPR Plant growth promoting rhizobacteria PCR Polymerase chain reaction RT-PCR Real time polymerase chain reaction PCA Principal components analysis TSB Trypticase soy broth TSA Trypticase soy agar WHO World health organization YEM Yeast extract manitol

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1. GENERAL INTRODUCTION

Iron (Fe) is an essential micronutrient for all living organisms, including human beings, but

it typically is inadequate in soil in the plant available form. Consequently, Fe deficiency is a

major human health and development problem worldwide. The World Health Organization

(WHO) has reported that approximately 3.7 billion people are affected by Fe deficiency and

nearly 2 billion among them are anaemic (Yang et al., 2007). Iron deficiency is the most

dominant nutrient deficiency worldwide and mostly prevalent in countries of South Asia (Zlotkin

et al., 2004). For instance, in Bangladesh about half of all children and 70 percent of all women

are anaemic (Ahmed et al., 2012). The primary cause of Fe deficiency in developing countries is

low quantity and quality of dietary Fe intake (Shaw and Friedman 2011; Bowler, 2010). A diet consisting of poor Fe sources is one major reason for inadequate Fe intake. Consequently, providing healthy foods, together with supplementation and food fortification can reduce Fe- deficiency in humans. Unfortunately, these strategies are not affordable for low income families from developing countries (Foo et al., 2004; Haas et al., 2005).

The high protein content in lentil and pea makes these pulses an important food source for low-income people (Iqbal et al., 2006; Patterson et al., 2009; Roy et al., 2010). Lentils are rich in essential micronutrients including Fe, Zn and proteins (Bhatty, 1988; DellaValle et al., 2013;

Johnson et al., 2013). Lentil can be a cash crop to the producers, increase and improve crop rotations and reduce the chemical fertilizer requirement (Tonitto et al., 2006). Biofortification with Fe in staple foods, such as lentil, provides a cost-effective solution to alleviate Fe deficiency

1 in developing countries (Bouis and Welch, 2010; Bouis et al., 2011; Khush et al., 2012).

Biofortification has been considered the “second Green Revolution” (Lynch, 2007). Therefore, biofortification of lentil and pea may close the gap between Fe intake and requirements, and hence alleviate Fe deficiency (Thompson and Meerman, 2010; Murgia et al., 2012).

Currently, the Saskatchewan pulse crop industry is the major supplier of lentil and field pea globally. At this time, Saskatchewan supplies pulses to more than 130 countries around the world

(FAOSTAT, 2013; McVicar et al., 2017). Areas within Saskatchewan are very suitable for lentil production. The cold temperature in the prairies provides excellent protection against pathogens and insects and lentil is well adapted for soils of prairie regions (Bailey et al., 2000). The two main classes of lentil are red and green; among them the red lentil is a major class produced worldwide, but the red type is also predominant in Western Canada (McVicar et al., 2017).

Pea is a shallow rooted crop. Moist Chernozemic soils with high organic matter are well suited for the crop; however, pea can be grown in Brown Chernozemic soils due to its drought resistant capability (Bailey et al., 2000; Adderley et al., 2006). Pulse crop adaptation trials indicated that pea can easily be adapted in all agro-ecological zones in Saskatchewan (Gan et al.,

2003). Pea has become an alternative crop for farmers, who choose to diversify crop production in Saskatchewan. Both yellow and green pea cultivars are grown in Saskatchewan; approximately 80% of them are yellow types (Watts, 2011). According to Warkentin et al.

(2007), CDC Meadow is the most widely grown field pea variety in Saskatchewan; it has become popular due to its high production, good quality seeds, mildew and lodging resistance.

The other widely grown pea varieties in Saskatchewan include CDC Golden, CDC Amarillo,

CDC Striker and CDC Dakota.

Plant growth promoting rhizobacteria (PGPR) comprise a wide range of soil bacteria, which grow in association with plants and stimulate the growth of their host by increasing nutrient solubility, uptake and enhancement in the plant (Çakmakçi et al., 2006). Some bacteria increase nutrient availability for plants in the rhizosphere by direct or indirect mechanisms (Glick, 1995).

Numerous studies have reported that some bacteria increase the Fe uptake in various crops

(Bhattacharya, 2010; Pii et al., 2015; Scagliola et al., 2016). Iron content has been significantly increased in wheat grain inoculated with Trichoderma asperellum (de Santiago et al., 2011). Red pepper treated with Methylobacterium oryzae increased Fe content (Kim et al., 2010).

Siderophore-producing bacteria (e.g., Pseudomonas putida) is a strong Fe-solubilizing PGPR for rice plant (Sharma et al., 2013). However, information on the interaction between siderophore- producing bacteria and pulse crops including lentil and pea is scarce. The focus of this study was to demonstrate the role of siderophore-producing bacteria on Fe uptake on selected lentil and pea cultivars grown in Saskatchewan.

Overall objectives

1) To isolate siderophore-producing bacteria from rhizosphere and endorhizosphere of lentil

plants grown in different Saskatchewan soils;

2) To assess the siderophore production and growth promotion capacity of bacterial isolates;

3) To evaluate the impact of inoculants on Fe content in different lentil and pea cultivars.

Overall hypotheses

1) The abundance of culturable Fe-chelating bacteria isolated from rhizosphere and

endorhizosphere of lentil (Lens culinaris L.) is soil dependent.

2) The success of inoculation with siderophore-producing bacteria is plant cultivar specific.

3) Inoculation of lentil (Lens culinaris L.) and pea (Pisum sativum L.) with siderophore-

producing bacteria enhance Fe content.

Organization of the Thesis

This thesis organized in a manuscript format consisting of six chapters. Chapter 1 describes a general introduction followed by the literature review in Chapter 2. Chapters 3, 4 and 5 are written as research articles, all of which begin with a preface to understand the objectives and make connections within the thesis. Isolation and identification of siderophore-producing bacteria and the siderophore production capacity of isolates in vitro are described in Chapter 3.

Chapter 4 is the continuation of the previous study, where high siderophore-producing isolates were inoculated using a single lentil cultivar as the test crop to assess their effect on plant Fe uptake. Isolates that resulted in increased Fe uptake in lentil subsequently were inoculated in different lentil and pea cultivars in a growth chamber experiment and the results are presented in

Chapter 5. Finally, Chapter 6 summarizes the results from Chapter 3, 4 and 5 to draw a conclusion and addresses the areas for future research related to this topic. Articles that were cited in this thesis are listed in Chapter 7. Appendices are attached to the end of the document.

2. LITERATURE REVIEW

2.1 Importance of iron in human animal and plant

Iron (Fe) is an essential micronutrient for health and development of humans, animals and

plants as it is required for many metabolic processes such as oxygen transportation and cellular

respiration. Additionally, Fe is a component of hemoglobin, myoglobin, cytochromes and

enzymes (Soetan et al., 2010). Iron is the most important cofactor for the production of red blood

cells, conversion of blood sugar to energy and also plays an important role in the production and

functioning of various enzymes, essential biomolecules like amino acids, hormones and

neurotransmitters (Trumbo et al., 2001). Iron also is needed for white blood cells to help develop

the human immune system. In contrast, lack of Fe reduces red blood cell production and thus

causes anaemia both in humans and animals. For example, Chakravarty and Ghosh (2000)

reported that severe anaemia has a direct influence on maternal and child mortality. Additionally,

Fe deficiency is also implicated in metabolic processes that can cause neurological and behavioral disorders (Youdim et al., 1989; Beard, 1999).

In plants, Fe is essential for chlorophyll biosynthesis, to maintain the structure of

chloroplasts, nitrogen fixation, DNA replication, reactive oxygen species (ROS) scavenging, and

to act as an electron transporter (Braun et al., 1998; Briat et al., 2010). Iron also regulates plant

development and reproduction reactions including respiration, photosynthesis, reduction of

nitrates and sulphates. Legumes need a large quantity of Fe for nitrogen fixation in their nodules

(Burton et al., 1998). Iron deficiency can reduce chlorophyll synthesis resulting in chlorosis,

mostly found in young leaves. Iron (Fe) deficiency can reduce plant yield. For example, soybean

(Glycine max L. Merr.) is susceptible to Fe deficiency and, as a result, the production of this crop

is challenging in many regions due to Fe deficient soils (Naeve, 2006). Iron deficiency can reduce root growth and development (Li et al., 2008). Therefore, Fe deficiency can reduce crop

yield quality and quantity.

Adequate quantities of Fe are very important for proper growth and development of humans,

animals and plants. Plants can take up available Fe from soil, but the availability of Fe depends

on many factors including, pH, water, and microbial activity (Lindsay and Schwab, 1982;

Stockdale et al., 2013).

The heme Fe and non-heme Fe are the two major dietary sources of Fe for human and

animal. Heme Fe is mainly animal based (e.g., egg, yolk, red meat) whereas non-heme is mainly

plant based (e.g., green leaved vegetables (spinach), soybean, lentils, chickpeas, kidney beans)

(Carpenter and Mahoney, 1992; Kongkachuichai et al., 2002).

In many developing countries, human beings suffer from low Fe uptake from heme sources

due to the high price of animal products and/or traditional or cultural restrictions (La Frano et al.,

2014; Zielińska-Dawidziak, 2015). Most livestock also exhibit low heme Fe intake because

forages and cereal crops are the major source of dietary Fe for them (Vasconcelos and Grusak,

2006). For these reasons, plants are an important source of Fe for most humans and animals. The

increase in Fe content of plants ultimately improves human and animal health.

2.2 Availability of Fe for the plants in soil

Iron is the fourth most abundant element in the earth’s crust. Iron is present in most rocks as

primary minerals in Fe-magnesium silicate forms such as olivine, augite, hornblende, or biotite

(Chester and Hughes, 1967). Primary soil minerals are not stable in the soil and are weathered

slowly by water and oxygen, to release Fe (II) and Fe (III) ions. Iron content in the soil solution

is controlled and inversely proportional to the soil pH. Iron concentration reaches a minimum at

pH values between 7.4 to 8.5, which corresponds to the pH of most of the agricultural soils in the

world (Lindsay and Schwab, 1982). Plants need 10–4 to 10–8 M Fe (III) ions for normal growth,

but, only 10–17 M is soluble at pH 7.0 (Mori, 1999). Furthermore, Fe solubility is also regulated

by several factors such as the redox conditions of the soil, the presence of organic chelates (i.e.,

bacterial siderophores) that increase Fe availability in the soil solution (Neilands and Leong,

1986). Plant root exudates or humic substances can also increase Fe solubility (Robin et al.,

2008).

Iron is an important redox-active metal for plants. Iron is involved in nitrogen assimilation,

photosynthesis, respiration, hormone biosynthesis (e.g., ethylene, gibberellic acid),

osmoprotection and antipathogen (Hänsch and Mendel, 2009). Iron is also a major component of

chloroplasts.

Briat (2007) reported the importance of Fe uptake and homeostasis in legumes due to the

richness of their seed Fe content compared to cereals. Legumes and other dicotyledonous plants

take up Fe through Fe3+ reduction and Fe2+ transportation (Dudeja et al., 1997). However, legumes have a symbiotic relationship with Rhizobium bacteria present in their nodules to fix

nitrogen. Iron-containing nitrogenase and leghemoglobin proteins are very important for this

process (Tang et al., 1990). Iron uptake by the bacteroides is a potential Fe uptake mechanism

for legumes, e.g., Fe in soybean seed could be derived from nodules (Burton et al., 1998).

2.3 Mechanisms of Fe-chelation by crop associated bacteria in soil

Plant uptake of Fe can be enhanced through the presence of siderophores and subsequent chelation of Fe (Cline et al., 1982; Neilands and Leong, 1986). Kraemer (2004) reviewed the mechanisms by which siderophores solubilize Fe from Fe-bearing minerals and reported that siderophores can transport Fe from the soil solution to the surface of cells by a diffusion gradient. Additionally, siderophores are also capable of solubilizing organic Fe (e.g., Fe- containing humic acid). Along with organic matter, plant detritus are important source of Fe; siderophores can solubilize Fe form organic sources and return it in plant biomass before the formation of inorganic Fe minerals (Crowley and Kraemer, 2007).

Römheld and Marschner (1986) reported that phytosiderophores play a major role in preventing Fe-deficiency in grass species. However, microbial siderophores (low molecular chelating compounds) are very important in Fe nutrition in leguminous plants (Crowley et al.,

1991). Moreover, microbial siderophores can solubilize Fe (III) from insoluble Fe sources in the

rhizosphere (Jin et al., 2008). Studies using radiolabelled microbial siderophores suggested that

siderophores were used as a sole source of Fe by many plants including sorghum, oats, peanut,

cotton, cucumber, maize and sunflower (Nambiar and Sivaramakrishnan, 1987; Walter et al.,

1994; Dudeja et al., 1997; Stintzi et al., 2000; Dertz et al., 2006). Several authors including

Miethke and Marahiel (2007) and Mishra et al. (2014) suggested that microbial production of

8 siderophores in nodulated legumes in field conditions might increase the uptake of Fe from the soil. Many microorganisms produce siderophores in the rhizosphere and increase Fe solubility; hence these rhizosphere organisms increase plant Fe uptake (Crowley et al., 1991).

2.4 Siderophores

Siderophores are low molecular weight (500 – 1500 daltons) organic compounds, that are synthesized by many microbes to chelate Fe3+ from insoluble Fe compounds when growing under Fe deficient conditions (Miethke and Marahiel, 2007). There are over 500 different siderophores, of which the chemical structure of 270 siderophores have been identified

(Wandersman and Delepelaire, 2004; Hider and Kong, 2010). Siderophores can be categorized mainly into three types based on the chemical characteristics of the functional group or groups used for Fe3+ binding. These types are the catecholates/phenolates, hydroxamates and carboxylates (Miethke and Marahiel 2007).

The hydroxamate type of siderophores contain a hydroxamate group and each hydroxamate group provides two oxygens, which form a ligand with Fe3+ resulting in a hexadentate octahedral complex for each siderophore. The hydroxamate group comes from amines such as ornithine or lysine (Hider and Kong, 2010). Hydroxamate siderophores are produced by both fungi and bacteria and several of these siderophores include alcaligin, aerobactin, rhizobactin, and schizokinen desferrioxamine B (Crosa and Walsh, 2002).

The second most common type of siderophores are catecholates/phenolates siderophores, which are usually produced by bacteria (Vala et al., 2006). The catecholates/phenolates comprise mono or dihydroxybenzoic acid residues, which bind to Fe3+ and mostly originate from 2, 3- dihydroxybenzoic acid. Examples of catechol type siderophores include enterobactin and

vibriobactin whereas the phenolate type siderophores includes yersiniabactin and pyochelin

(Miethke and Marahiel, 2007; Hider and Kong, 2010).

The carboxylate type of siderophores is comprised of carboxyl and hydroxyl functional

groups, and these groups release two oxygens that bind to Fe3+ (Drechsel et al., 1995). The

rhizobactin DM4 is the first representative of the carboxylate class of siderophores; this is the

only siderophore commonly found in bacteria, archaea, and eukarya. Additional examples of other siderophores belonging to this group include staphyloferrin A and B, vibrioferrin, and rhizoferrin (Dave et al., 2006; Miethke and Marahiel, 2007).

In addition to these types, a fourth type of siderophore has also been identified and named

“Mixed type”, which are a combination of any of the above types of siderophores. Some examples of this type of siderophores include heterobactin, mycobactin T, and aerobactin

(Miethke and Marahiel, 2007).

2.5 Effect of siderophore-producing bacteria on Fe nutrition and growth promotion of crops

Most of the plant and root associated bacteria originate from the bulk soil. Bacteria may move to the rhizosphere and then within the rhizoplane of their host plants before exhibiting potential beneficial effects (Germida et al., 1998; Compant et al., 2010). Some rhizoplane bacteria can also enter into plant roots, and some may move to the plant shoot (Hardoim et al.,

2008).

Several researchers including, de Freitas and Germida (1992) and Mia et al. (2010) have explored beneficial effects of rhizosphere bacteria on agricultural crops. Bacteria exhibit various

mechanisms in soils by which they can assimilate atmospheric nitrogen, co-operate with plants

for growth promotion, detoxify potentially toxic chemicals and/or cause plant diseases, degrade

plant detritus. Rhizosphere bacteria can effectively colonize plant roots and improve plant

nutrient uptake by improving the root system and/or by solubilizing insoluble nutrients (López-

Bucio et al., 2007). This is a potential method by which the need for chemical fertilizers use could be reduced, leading to sustainable organic agriculture (Requena et al., 1997).

A number of studies of the rhizosphere populations of various plant species have revealed that Pseudomonas spp. were the predominant siderophore-producing bacteria in the root zone

(Chaiharn et al., 2009). Additionally, Pseudomonas isolated from rhizosphere and endorhizosphere of amaranth (Amaranthus sp. L.) and garlic (Allium sativum) root, respectively, increased Fe content in lentil grown in potted soil in a greenhouse study (Mishra et al., 2011).

Similarly, Sharma and Johri (2003 a, b) also reported that siderophore-producing Pseudomonas spp. were able to increase Fe content and promote growth of mung bean (Vigna radiata L.

Wilzeck) and maize (Zea mays L.) plants grown under Fe-limiting conditions. Conversely, Fe content decreased in both maize and pea treated with siderophore of a Pseudomonas sp., strain

B10 grown in a sterile nutrient solution, thus indicating that depending on the growth conditions, such as lack of competition with the native microflora, some Pseudomonas also may affect plant growth negatively (Becker et al., 1985).

In addition, to promoting plant growth and yields, beneficial siderophore-producing bacteria are increasingly being used as bio-control agents (Haas and Défago, 2005; Höfte and Altier,

2010; Wensing et al., 2010; Yu et al., 2011). Bacterial siderophores play an important role in

suppressing plant pathogenic fungi including Fusarium and Pythium spp. by generating Fe- limiting conditions to the pathogens.

2.6 Isolation of siderophore-producing microorganisms

Plant rhizosphere and endorhizosphere bacteria can be isolated on specific growth media including the universal Chrome Azurol S (CAS) medium for the production of Fe-chelating substances (Machuca and Milagres, 2003; Louden et al., 2011; Gamit and Tank, 2014). The CAS have very high attraction for iron (III). Chrome Azurol S, iron (III) and hexadecyl-trimethyl- ammonium-bromide forms a blue colour complex solution and serves as an indicator. When the

Fe removed from the solution by a chelator, the blue color solution turns to orange. Siderophore characterization and determination can be achieved using the supernatants of liquid culture. This technique is also appropriate for agar plates. Orange halos around the colonies on CAS agar plates are an indication of siderophore production. The method was successfully used by Schwyn and Neilands (1987) to screen different strains of Escherichia coli and Rhizobium meliloti in the high affinity Fe uptake system. Overlaid Chrome Azurol S (O-CAS) medium is also used in many studies to detect siderophore (Pérez-Miranda et al., 2007; Qing-Ping Hu and Xu, 2011).

Modified CAS can also be used to measure siderophore production produced by fungi (Machuca and Milagres. 2003).

2.7 Methods to measure siderophore production

Several methods are available to measure microbial siderophore production in vitro. Among them, the Chrome Azurol S (CAS) assay method is commonly used (Alexander and Zuberer,

1993). This assay is very useful when screening for siderophore production both in culture

supernatants and in agar plates. Alexander and Zuberer (1991) used modified CAS assay solution

to measure siderophore in liquid culture. Deferoxamine mesylate was the standard solution in

microtiter methods.

Additional methods commonly used to measure siderophores include (i) the Arnow's

method. It uses 1 mL culture supernatant, which is mixed with 1mL 0.5 N HCl followed by 1 mL

nitrate molybdate (sodium nitrite 10 g and sodium molybdate 10 g were dissolved in 100 mL

distilled water to made nitrate molybdate) 1mL 1N sodium hydroxide and final volume was

adjusted to 5 mL with deionized distilled water. In this assay, 2, 3-di-hydroxybenzoic acid is

used as a standard and absorbance measured at 510 nm. Use of Arnow's method has allowed

detection of siderophores in different species of bacteria (Carson et al., 1992; Jikare and Chavan,

2013). In fact, Tailor and Joshi (2012) successfully used the Arnow's method to measure

siderophores in Pseudomonas fluorescens strain isolated from rhizosphere of sugarcane. In the

Csaky’s method, method, culture supernatants hydrolyse with 3N sulfuric acid followed by

neutralization with 35% anhydrous sodium acetate. Hydroxylamine hydrochloride was used as a

standard. Jikare and Chavan (2013) measured siderophore in culture supernatants of Bacillus

shackletonii bacteria (isolated from rhizosphere of groundnut) using the Csaky’s method and

found that Bacillus shackletonii are able to produce siderophore. In the third method, radiotracers

e.g., radioactive iron (59Fe) have also been used to measure microbial siderophore in culture

59 59 supernatants of a Bradyrhizobiur strain and found that siderophore can bind Fe form FeCl3;

(Nambiar and Sivaramakrishnan, 1987). A fourth method is High Performance Liquid

Chromatography Mass Spectrometry (HPLC-MS) (Essén et al., 2006). Briefly, soil is collected, centrifuged and filtered, and ferric Fe is added to the filtered solution to allow formation of Fe- complexes. Then, siderophore in the soil solution is measured by HPLC-MS using supernatants.

A fifth method is SideroTec® assay kit (Gi et al., 2015). In this method, the supernatant is mixed

with assay solutions in a microtiter plate and absorbance measured at 630 nm with a microplate

reader. Gi et. al. (2015) used the kit to study siderophore production in culture supernatants of

Pseudomonas aeruginosa.

From the above described methods, it was concluded that the SideroTec® assay kit method is

rapid, reliable and straight forward, hence it was adopted in the current thesis research to study siderophore production.

2.8 Methods for identification of the microbes

Culture dependent and culture independent methods have been used by researchers to

analyze microorganisms (Ros et al., 2009). Culture dependent methods are based on isolation of

microorganisms using wide variety of culture media and their phenotypic characteristics i.e. size,

shape, elevation etc. Culture media composition are prepared to maximize the diverse microbial

groups or specific group of microorganisms. For example, Trypticase soya agar is used to isolate

total heterotrophic bacteria, where as Kings B, Yeast Extract Manitol (YEM) and Chrome

Azurol S (CAS) medium are specific for the isolation of pseudomonads, rhizobia and siderophore-producing bacteria, respectively. Based on utilization of carbon sources, a Biolog- method has also been introduced, but the functional diversity of microbial community is not represented accurately in this process (Topp, 2003). Only a small fraction of the microbes in soil are accessible to study using culture dependent methods, however, recent studies indicated that this percentage can be increased significantly by using different cultivation procedures (Hill et al., 2000).

Current developments in molecular based methods have aided a rapid identification of soil

microbial communities. Soil microbial community structure can have measured by assessing

culture-independent Phospholipid fatty acid analysis Phospholipid fatty acid (PLFA) analysis.

Phospholipid fatty acid (PLFA) analysis also have been used to measure gross changes in soil

quality as a result of different agronomic and soil management practices (Xue et al., 2013;

Watzinger, 2015).

Phospholipid fatty acids are present in all living cells and are potentially useful signature

molecules. Unique fatty acids are representative to specific groups of organisms. Specific fatty

acid methyl esters (FAMEs) have been used as a recognised taxonomic discriminator for species

identification. The occurrence of unique fatty acids in soil discloses the presence of specific

organisms or groups of organisms in which those fatty acids can be found.

Polymerase Chain Reaction (PCR) or Real Time Polymerase Chain Reaction (RT-PCR)

based molecular techniques are generally specific or generic targets for DNA or RNA of soil and

plant microorganisms. The 16S and 18S ribosomal RNA (rRNA) or their genes (rDNA) serve as suitable biological markers for eukaryotes and prokaryotes, respectively. The 16S or 18S rDNA of all PCR-accessible species present in the soil or plant yielded a mixture of DNA called PCR product with the help of primers. The mixed PCR products can be used for microbial community fingerprinting techniques including Denaturing Gradient Gel Electrophoresis (DGGE) and gene sequencing.

Polymerase Chain Reaction (PCR) based DGGE is probably the most common method used to study microbial communities in environmental samples. Group-specific PCR-DGGE have been used to analyze specific subgroups of complex environmental communities. Group-specific

PCR-DGGE methods are also now available for studying prokaryotic groups such as

Actinomycetes, Bacillus, Paenibacillus, Pseudomonas, the α- and β-Proteobacteria, ammonia- oxidizing bacteria, and N2-fixing bacteria. Methods for other specific groups are under development. In several studies, molecular techniques based on the 18S rRNA gene have been applied to assess the fungal diversity in soil (Smit et al., 1999; da Silva et al., 2003; Anderson and Cairney, 2004).

The most promising and reliable strategy for the classification and identification of bacteria includes the analysis of the 16s rRNA. The 16s rRNA comparison with published data (NCBI,

GenBank) apparently confirm close relatives of the strain. Chemotaxonomic methods can be applied to detect similarities to existing taxa and describe the features of the organism. For example, if closely related sequence of the 16s rRNA detected between isolates, more detailed information can be found by polar lipid and fatty acid patterns analysis.

3. ISOLATION AND IDENTIFICATION OF SIDEROPHORE-PRODUCING

BACTERIA FROM RHIZOSPHERE AND ENDORHIZOSPHERE OF LENTIL PLANTS

GROWN IN DIFFERENT SASKATCHEWAN SOILS

3.1 Preface

In this study, siderophore-producing rhizospheric and endorhizospheric bacteria associated with lentil plants grown in different Saskatchewan soils were isolated from plants cultivated under growth chamber conditions. Bacteria were isolated from roots using culture dependent methods to establish a siderophore-producing bacteria culture collection. Prior to selection and identification of candidate isolates for further experiments, siderophore production capacities were assessed in vitro.

3.2 Abstract

Siderophore-producing bacteria can colonize both in rhizosphere and endorhizosphere. Prior to isolating siderophore-producing bacteria, lentil cv. CDC Milestone was grown in a growth chamber study using eight different agricultural soils from Saskatchewan. Plants were harvested at 45 days after planting (DAP) and roots were used to isolate root associated bacteria.

Siderophore-producing and total heterotrophic bacteria were isolated on Chrome Azurol S and

Trypticase soy agar medium, respectively. Populations of total siderophore-producing and heterotrophic bacteria were significantly different among soils from distinct locations. As

expected, bacteria populations were significantly higher in the rhizosphere when compared to the endorhizosphere counterparts and the percentage of siderophore-producing bacteria were much lower when compared to total heterotrophic populations. Lentil plants grown in Elrose soil exhibited the highest population and percentage of siderophore-producing bacteria in both in the rhizosphere and the endorhizosphere and isolates produced siderophores ranging 0.04 to 119.26

µg∙mL-1 in vitro. Total-Fe uptake by plants were also significantly different among soils from different locations, however, this difference did not correlate with soils’ total Fe content. In this

study, the top 68 siderophore-producing isolates were identified using Sanger sequencing method

(16S rRNA gene sequencing). Basic Local Alignment Search Tool (BLAST) analysis revealed a

wide range of species, which belonged to the following families: Bacillaceae, Burkholderiaceae,

Enterobacteriaceae, Oxalobacteraceae, Pseudomonadaceae, Rhizobiaceae and

Xanthomonadaceae. Among the identified siderophore-producing isolates, the SPG and Elrose

soil exhibited a diverse group of siderophore-producing isolates followed by Laird, Sutherland,

Limerick, Skarsgard, Scott and Cabri soils.

3.3 Introduction

Soil is the primary source of many microorganisms including bacteria (Foster, 1988; Giri et

al., 2005) and soil bacteria is considered an important factor in agricultural crop production.

Culturable bacteria can be isolated from soil, plant rhizosphere and/or endorhizosphere using

universal non-selective cultural growth media such as Tryptic Soy, or selective media including

King’s B and Yeast extract-manitol (YEM), which are selective for Pseudomonas and Rhizobia,

respectively. An important mechanism by which soil microorganisms help plant growth includes

siderophore production (Cattelan et al., 1999; Joseph et al., 2007; Glick, 2012). When associated

with a crop, some bacteria increase Fe solubility for plant uptake and make Fe unavailable to

plant pathogens (Höfte and Altier, 2010). Furthermore, siderophore production is a well-known plant growth promotion mechanism that can be assessed in vitro on Chrome Azurol S (CAS)

medium (Radzki et al., 2013; Ahmed and Holmstrom, 2014). However, populations of siderophore-producing bacteria in soil are relatively low and, as a result, just a few rhizosphere bacteria can produce siderophore. In fact, Mishra et al. (2011, 2014) isolated rhizosphere bacteria from rice and reported that only a small subset of the isolates was able to produce siderophores.

Similarly, Chaiharn et al. (2009) and Husen (2003) isolated rhizosphere bacteria from bulk soil, rhizosphere and root exudates and reported that all isolates were not able to produce siderophore

in vitro.

Siderophore-producing bacteria can produce various amount of siderophores. For example,

Sharma and Johri (2003a) reported siderophore production levels associated with bacteria

ranging between 7 to 57 mg·L-1 in vitro.

It has been demonstrated that inoculation of siderophore-producing bacteria on various crops

has significant effects on growth promotion and nutrient uptake (Compant et al., 2005). Gamit

and Tank (2014) reported that seed inoculation with siderophore-producing rhizobacteria

significantly increased Fe uptake of pigeon pea (Cajanus cajan) and most of the bacteria were

identified as Pseudomonads. The use of rhizospheric bacteria as inoculants to enhance legume

crop growth is not new. For example, Rhizobium inoculants are very important in leguminous

crop production and are commercially available in the market (Date, 2000; Morel et al., 2012;

Bashan et al., 2014). Moreover, co-inoculation of Rhizobium leguminosarum with Pseudomonas sp. exhibited a maximum increase of 115.7% total Fe uptake in lentil plants (Mishra et al., 2011).

World annual lentil production is estimated at four million tons, of which approximately one

million tons is grown in Saskatchewan, Canada and Canadian lentils are exported to many

countries (FAOSTAT, 2013). For these reasons, lentil has become an important crop in

Saskatchewan agriculture industry. However, little is known about siderophore-producing

bacteria of leguminous crops, especially in Saskatchewan. The objectives of this study were to

isolate siderophore-producing bacteria associated with lentil using culture dependent methods

and assess their capacity to produce siderophore in vitro.

3.4 Materials and methods

3.4.1 Soil collection and growth chamber study

Soil was collected immediately after harvest from eight locations in Saskatchewan during

the 2013 growth season. Research plots where lentil had been being grown, located at Cabri,

Elrose, Laird, Limerick, Skarsgard, Scott, Sutherland and the Saskatchewan Pulse Grower (SPG)

land near Saskatoon, Saskatchewan, were chosen for soil sample collection. Approximately 10

kg of soil was collected from each location by excavating the surface to a depth of approximately

15 cm and subsequently air dried. Air-dried soil was passed through a 4-mm sieve prior to the experiment establishment. Soil subsamples were obtained and sieved (2-mm mesh) for physical/chemical analysis (Table 3.1). Soil (1.0 kg) was placed in replicated pots (three

replicates), which were lined with plastic bags to prevent loss of water and incubated at 80%

field capacity (FC) for two weeks in the growth chamber set at 25°C and 20°C day and night

temperature, respectively. Plastic bags were slightly closed to prevent excess evaporation. After

incubation, each pot was seeded with eight surface sterilized (70% [v·v–1] ethanol for 3 min, 1%

[v·v–1] sodium hypochlorite for 1 min and washed five times with sterile tap water) lentil (CDC

Milestone) seeds and four plants were kept in each pot after germination. Plants were grown in

the growth chamber at 16 h and 8 h day and night cycle, respectively. During the experiment,

plants were watered to 80% FC by weighing each pot. Pots were randomly repositioned once a

week to prevent edge effects in the growth chamber. Plants were harvested at the flowering stage

(45 DAP). Roots were separated from the bulk soil and used for the isolation of rhizosphere and

endorhizosphere bacteria.

3.4.2 Isolation of siderophore-producing and total heterotrophic bacteria from rhizosphere

and endorhizosphere using culture dependent method

The complex CAS solid medium used to isolate siderophore-producing bacteria was prepared as described by Alexander and Zuberer (1991). Briefly, the medium consists of four solutions, which were sterilized separately before mixing. Solution 1: 10 mM HCl was mixed

-1 with 1 mM FeC13·6H20 and added to the 50 mL CAS (1.21 mg·mL ) solution. This produced a dark purple solution. The resulting solution was added slowly to a 40 mL hexadecyl-trimethyl- ammonium-bromide (HDTMA) (1.82 mg·mL-1) solution with continuous stirring. The final solution was a dark blue colour. Solution 2: NaCI (0.5 g), KH2PO4 (0.3 g), NH4Cl (1.0 g) and

piperazine-N,N'-bis[2- ethanesulfonic acid] (PIPES) (30.24 mg) were added in 750 mL deionized

water under agitation until dissolved. Fifty percent [w·v–1] KOH was used to adjust the pH to

6.8, and the final volume of 800 mL was attained by adding deionized water. Agar solution was added before autoclaving.

Table 3.1 Physico-chemical properties of soil samples collected from different locations in Saskatchewan in 2013. All analyses were analyzed at ALS Environmental Laboratory, Saskatoon.

Location Soil zone Texture FC pH Organic NO PO K+ SO Total Fe matter (×103) + 3− 2− 3 4 4 mL∙kg-1 g∙kg-1 ------mg∙kg-1 ------

Cabri Brown Silty clay loam 441 7.43 34.2 10.1 14.6 1060 10.5 26.7

Limerick Brown Clay 427 7.43 26.3 10.8 8.9 766 8.3 32.2

Elrose Dark Brown Loam 289 6.33 27.4 14.8 21.7 458 7.2 18.9

Skarsgard Dark Brown Silt loam / Loam 288 5.91 39.5 9.8 48.4 621 23.2 14.9

22 SPG M Dark Brown Silt loam 375 5.62 59.3 17.1 30.8 546 7.5 17.6

Scott M Dark Brown Loam 280 5.78 35.5 3.5 43.4 401 6.7 14.2

Sutherland Dark Brown Loam 314 6.35 33.9 8.8 18.2 454 7.4 18.9

Laird Black Silt loam 472 6.06 90.9 24.9 76.7 990 13.8 17.0 ‡ Texture was measure by mini-pipet method; FC= field capacity ¶ pH measured in a 1:2 soil:water suspension (Hendershot et al., 2008) ǂ Organic matter (Walkley and Black, 1934)

⸸ N= CaCl2 extractable nitrate, NO3-N (Houba et al., 2000)

† P and K= Modified Kelowna extractable phosphate, PO4-P and K (Qian et al., 1994) § S= CaCl2 extractable sulphate, SO4-S (Houba et al., 2000) ‡‡ Total Fe was measured in strong acid digestion (Alcock, 1987) # SPG = Saskatchewan Pulse Grower

Solution 3: glucose (2 g), mannitol (2 g), MgSO4·7H2O (493 mg), CaCl2 (11 mg), CuSO4·5 H2O

(0.04 mg), ZnSO4·7H2O (1.2 mg), MnSO4·H2O (1.17 mg), H3BO3 (1.4 mg) and Na2MoO4·2H2O

(1.0 mg) were added in 70 mL water. Solution 1, 2 and 3 were autoclaved, cooled to 50°C.

Solution 4: Casamino acids (100 g·L-1) was mixed with 30 mL cooled autoclaved water.

Solution 4 was added using a sterile filter to Solution 3 and Solution 2 very carefully to avoid air bubbles. Solution 1 was added in the end, with slow stirring to mix the ingredients without foam formation. All solutions were freshly prepared with deionized water for each batch of CAS medium. Glassware used in CAS media preparation were kept overnight with 3 M HCl and rinsed with deionized water to remove any metal traces.

Rhizospheric bacteria were isolated using the procedure described by Siciliano and Germida

(1999). Briefly, 2.0 g of root containing rhizospheric soil was placed in a 500 mL Erlenmeyer flask containing 200 mL of sterile phosphate buffered saline (PBS). Then, the roots were shaken on a rotary shaker (150 rpm) for 25 min. The resulting slurry was serially diluted in PBS and 0.1 mL of the appropriate dilutions spread onto agar plates (n=3). Specifically, siderophore- producing and total heterotrophic bacteria were isolated from lentil roots on Chrome Azurol S

(CAS) medium (Fig. 3.1) and 1/10 strength Trypticase Soy Agar (TSA), respectively (Alexander and Zuberer, 1991; Siciliano and Germida, 1999). Cycloheximide (50 mg.L–1) was added to TSA medium as a fungistatic agent. Colony-forming units (cfu) were counted in each plate after 3 d and 7 d for total heterotrophs and siderophore-producing bacteria, respectively. For the isolation of endorhizosphere bacteria, 1.0 g of surface sterilized washed roots (Siciliano and Germida,

1999) were transferred into Erlenmeyer flasks containing 100 mL PBS amended with NaClO

(1.05% [v.v–1]). Then, the roots were agitated at 150 rpm for 15 min and washed 10 times with

100 mL sterile tap water to eliminate traces of bleach. To confirm the success of the surface 23

sterilization procedure, 0.1 mL of the final wash was spread onto TSA plates and incubated at

28°C. Then, the roots were cut in small pieces and triturated aseptically using a sterile mortar and pestle with 10 mL of PBS.

Fig. 3.1 Isolation of siderophore-producing bacteria on Chrome Azurol S (CAS) medium.

Orange halo represents the production of siderophore.

The resulting solution was serially diluted in sterile PBS and 0.1 mL of the appropriate dilutions spread on TSA and CAS plates (n=3), as described above. Single colonies (n=3) of candidate siderophore-producing bacteria isolated on CAS were selected based on morphological characteristics including; shape, size, elevation and motility. Purity was assessed by streaking individual colonies on TSA media. Purified strains were stored in a 1:1 mixture (v·v–1) of triptic soy broth (TSB) and glycerol at –80°C (Sharma and Johri, 2003b; Ythier et al., 2012).

3.4.3 Measuring siderophore production capacity of isolated root associated bacteria in

vitro

Siderophore production by individual isolates was measured in vitro. Quantitative

measurement of siderophore production by each candidate was assessed using a SideroTec®

Assay kit (Maynooth, Ireland; http://www.emergenbio.com), according to the manufacturers’

instructions. This is a modified Chrome Azurol S (CAS) method. A strong Fe chelator such as

desferoxamine removes the Fe from the dye complex and change the color from blue to orange

(Marques et al., 2012). Turbidity or other disturbance in the well may result in a failure to

accurately measure absorbance for each well by the colorimeter. The method is based on the

change of color and the actual causes of color changes may not be identified by this method. All

isolates were tested for the Fe chelation using SideroTec® Assay kit. Briefly, isolates were grown in 10 mL of Fe free minimal media 9 (M9) for 72 h at 28°C with constant shaking at 120 rpm. After incubation, a 1.5 mL aliquot of liquid culture was centrifuged (4000 × g for 2 min, three times) and siderophore concentration in the cell free supernatant was measured. Briefly, a

100 µL aliquot of supernatant was mixed with 100 µL of the assay solution. After 10min the absorbance was measured at 630 nm with a microplate reader. A standard curve was created using known concentrations of siderophore provided with the kit. Isolates exhibiting superior siderophore production were tested further for the ability to enhance Fe uptake in lentil.

3.4.4 Identification of siderophore-producing bacteria

Isolates that exhibited superior siderophore production were identified. Isolates were grown

in liquid culture (TSB) for 48 h and then stored in 50% (v·v-1) glycerol stock overnight prior to identification. Glycerol stocks were sent to Genome Quebec, Montreal, for sequencing. Identities were established by comparison of the sequences with the National Center for Biotechnology

Information (NCBI) GenBank databases.

3.4.5 Statistical analysis

The growth chamber studies were conducted in a completely randomized design. Bacterial cfu data were transformed using Log10 (Loaces et al., 2011). The differences between treatments

were analyzed using factorial ANOVA at a 5% significant level. The post hoc analysis (Tukey’s

test) was used to separate means using SAS software, version 9.4 (SAS Institute Inc., Cary, N.C.,

USA). Box plot and correlation coefficient were conducted using R software, version 3.3.0 (R

Core Team, 2016, R Foundation for Statistical Computing, Vienna, Austria).

3.5 Results

3.5.1 Abundance of culturable siderophore-producing bacteria

Total culturable siderophore-producing (Fig. 3.2) and heterotrophic (Fig. 3.3) bacteria

differed between in rhizosphere and endorhizosphere of lentil roots. The population of total

siderophore-producing-bacteria ranged from 3.91 to 6.25 log cfu·g-1 (fresh root weight) and

2.42 to 4.17 log cfu·g-1 in the rhizosphere and endorhizosphere, respectively (Fig. 3.2). The

abundance of total heterotrophic bacteria in the rhizosphere and endorhizosphere ranged from

6.90 to 8.05 log cfu·g-1 (fresh root weight) and 5.21 to 6.67 log cfu·g-1 respectively (Fig 3.3). A

significantly higher total culturable heterotrophic population was detected in the rhizosphere of

lentil grown in Elrose (8.03 log cfu·g-1) and Laird (8.05 log cfu·g-1) soil compared to

Sutherland, Scott and Skarsgard, whereas a significantly higher total population in

endorhizosphere was observed in lentil grown in Skarsgard (6.67 log cfu·g-1) and Limerick

(6.45 log cfu·g-1) soils compared to the other soils. Plants grown in Elrose soil exhibited a

significantly higher population of siderophore-producing bacteria in both rhizosphere (6.25 log

cfu·g-1) and endorhizosphere (4.17 log cfu·g-1) (Fig. 3.2) compared to the other soils, except

Laird and Skarsgard, respectively. As expected, rhizosphere populations of siderophore- producing bacteria were significantly higher than in the endorhizosphere in all plants grown in all soils studied.

9 8

7 a 6 b ab b root)) root)) b 1 - cd 5 cd def cde 4 g gh g efg fg 3 hi i 2 1 Population (log (cfu g 0 Sutherland Cabri Laird Limerick Scott Skarsgard SPG Elrose Soil Locations Fig. 3.2 Abundance of culturable siderophore-producing bacteria (colony forming unit= cfu) in selected Saskatchewan soils assessed on Chrome Azurol S medium. Different letters indicate significant differences using Tukey’s post hoc test at α=0.05. 27

Lentil plants grown in Elrose soil exhibited the highest percentage of siderophore-producing bacteria in both in the rhizosphere and the endorhizosphere compared to the other soils (Fig. 3.4).

As expected, when compared to total heterotrophic population, the percentage of siderophore- producing bacteria were very low (Fig. 3.4), and ranged from 0.05 to 1.66 % and 0.09 to 2.05 % in the rhizosphere and endorhizosphere, respectively.

9 ab ab a ab a 8 cd bc e de

1 root)) de

- fg 7 fg fg fg f 6 g 5 4 3

Population (log (log Population (cfu g 2 1 0 Sutherland Cabri Laird Limerick Scott Skarsgard SPG Elrose Soil Locations Fig. 3.3 Abundance of culturable total heterotrophic bacteria (colony forming unit = cfu) in selected Saskatchewan soils assessed on Trypticase soy agar (TSA) medium. Different letters indicate significant differences using Tukey’s post hoc test at α=0.05.

2.50 Rhizosphere Endorhizosphere 2.00

1.50

1.00

0.50

Siderophore producing bacteria (%) bacteria producing Siderophore 0.00 Sutherland Cabri Laird Limerick Scott Skarsguard SPG Elrose Soil Locations

Fig. 3.4 Percentage of siderophore-producing bacteria in selected Saskatchewan soils in relation to total heterotrophs.

Plants grown in soils from SPG and Laird exhibited significantly higher shoot weight (3.87 g·pot-1 and 3.59 g·pot-1, respectively) compared to the other soils (Table A.2). In contrast, the lowest plant biomass (1.66 g·pot-1) was observed in plants grown in Sutherland soil. Similarly, a

significantly higher total Fe content was detected in plants grown in soil from SPG

(267 µg·pot-1), but the soil from Limerick yielded plants with the lowest Fe content (i.e., 68

µg·pot-1) (Table A.2). In the current study, total Fe content and shoot weight in host plant did not correlate with the total Fe in soil (data not shown).

3.5.2 Siderophore production by individual isolates

A total of 491 siderophore-producing bacteria, including 263 and 228 from the rhizosphere and endorhizosphere, respectively, were isolated on CAS medium from lentil roots grown in eight different soils (Fig. 3.6). Lentil plants grown in Limerick soil yielded the highest number

(n=87) of potential siderophore-producing candidate bacteria including 38 and 49 isolates from endorhizosphere and rhizosphere, respectively, compared with the plants grown in the remaining soils.

All 491 siderophore-producing bacteria were grown in a Fe free M9 minimal medium prior to testing for siderophore production. In the current study, some isolates did not regrow and siderophore production was not detected in some isolates; however, 462 isolates were able to produce siderophores that were measured successfully using SideroTec® kit (Fig. 3.5).

Standard Unknown

Fig. 3.5 Measuring siderophore concentration using SideroTec® Assay kit. Standard solution came with the kit.

Bacterial growth measured by Klett readings of some isolates grown on Fe free M9 minimal

medium indicated that bacterial growth did not correlate with siderophore production (Fig. A.1).

The siderophore-producing bacteria were capable of producing siderophores in concentrations

ranging from 0.04 to 119.54 µg∙mL-1 (Table A.2). Of these, only 13 isolates exhibited relatively

high siderophore production, that is, >50 µg∙mL-1 (Fig. 3.6). The most effective and ineffective siderophore-producing isolates were SPG2R11 and SPG3R12, which produced 119.54 µg∙mL-1

and 0.04 µg∙mL-1, respectively. Interestingly, both bacteria were isolated from lentil grown in the same soil.

the interquartile range. Values more than 1.5 times the interquartile range are presented as dots (●), rhizosphere and endorhizosphere isolate numbers (n) were included for each location.

3.5.3 Identification of siderophore-producing bacteria

Siderophore-producing isolates were sent to Genome Quebec, Montreal for sequencing.

Sequencing was performed using Sanger sequencing method (16S rRNA gene sequencing). A

total of 68 isolates were identified successfully by comparing the results with the gene bank database of National Center for Biotechnology Information (NCBI) (Table 3.2) and using the

Basic Local Alignment Search Tool (BLAST) (Johnson, 2008). Analyses revealed a wide range of siderophore-producing bacteria which belonged to various families including Bacillaceae

(n=3), Burkholderiaceae (n=23), Enterobacteriaceae (n=3), Oxalobacteraceae (n=4),

Pseudomonadaceae (n= 25), Rhizobiaceae (n=9) and Xanthomonadaceae (n=1). Gene

sequencing analyses, indicated that isolate SPG2R11, the bacteria that produced the greatest

levels of siderophore, was identified as Herbaspirillum huttiense strain NBRC 102521 and

belongs to the Oxalobacteraceae group with 99 percent gene sequence similarity.

Herbaspirillum huttiense strain NBRC 102521 was only found in the rhizosphere of lentil grown

in SPG soil and was the only isolate from Oxalobacteraceae group identified in the current study. Rhizobium nepotum Strain 39/7 was only found in the endorhizosphere, whereas

Rhizobium naphthalenivorans strain TSY03b, was found only in rhizosphere of lentil. All siderophore-producing isolates from Pseudomonadaceae group were commonly found in both

rhizosphere and endorhizosphere. Among the identified isolates, the SPG and Elrose soil had

very diverse populations of siderophore-producing isolates followed by Laird, Sutherland,

Limerick, Skarsgard, Scott and Cabri soils (Table 3.2)

Table 3.2 Ranking of siderophore-producing isolates and identification according to Sanger (16S rRNA gene) sequencing.

Rank of Siderophore† Origin Isolate location Isolate siderophore Group Closest NCBI‡ relative Similarity(%) (µg·mL-1) production SPG Rhizosphere SPG2R11 120 1 Oxalobacteraceae Herbaspirillum huttiense strain NBRC 102521 99 SPG Rhizosphere SPG2R12 105 2 Oxalobacteraceae Herbaspirillum huttiense strain NBRC 102521 100 SPG Rhizosphere SPG3R9 101 3 Oxalobacteraceae Herbaspirillum huttiense strain NBRC 102521 99 Scott Rhizosphere Sc2R3 77 4 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 100 Scott Rhizosphere Sc2R2 68 5 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 99 Limerick Endorhizosphere Li3E21 67 6 Pseudomonadaceae family Pseudomonas brassicacearum NFM421 100 Scott Rhizosphere Sc2R1 66 7 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 99 Limerick Rhizosphere Li1R8 64 8 Pseudomonadaceae family Pseudomonas brassicacearum NFM421 99 SPG Rhizosphere SPG2R4 64 9 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 99 Laird Endorhizosphere La3E13 60 10 Pseudomonas fluorescens group Pseudomonas mandelii strain NBRC 103147 99 Limerick Rhizosphere Li1R7 52 11 Pseudomonadaceae family Pseudomonas brassicacearum NFM421 100 SPG Rhizosphere SPG2R6 51 12 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 100 Scott Rhizosphere Sc2R12 48 13 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 99 Elrose Endorhizosphere El3E13 45 14 Rhizobium/Agrobacterium group Rhizobium nepotum strain 39/7 100 Elrose Rhizosphere El1R5 45 15 Bacillus aryabhattai Bacillus aryabhattai strain B8W22 97 33 Scott Rhizosphere Sc2R10 44 16 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 100 SPG Rhizosphere SPG1R1 42 17 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 99 Scott Rhizosphere Sc2R11 40 18 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 99 SPG Rhizosphere SPG2R9 40 19 Rhizobium/Agrobacterium group Rhizobium naphthalenivorans strain TSY03b 100 SPG Rhizosphere SPG1R2 38 20 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 100 Sutherland Rhizosphere Su2R5 38 21 Pseudomonas fluorescens group Pseudomonas mandelii strain NBRC 103147 99 Skarsguard Rhizosphere Sk3R2 37 22 Pseudomonas fluorescens group Pseudomonas mandelii strain NBRC 103147 99 SPG Rhizosphere SPG1R3 37 23 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 100 Cabri Rhizosphere Ca2R1 37 24 Pseudomonadaceae family Pseudomonas brassicacearum NFM421 99 Laird Rhizosphere La3R3 36 25 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 99 Laird Rhizosphere La3R2 33 26 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 99 Limerick Endorhizosphere Li3E19 33 27 Pseudomonadaceae family Pseudomonas brassicacearum NFM421 100 Elrose Rhizosphere El1R8 33 28 Burkholderiaceae family Burkholderia terricola strain LMG 20594 100 Skarsguard Rhizosphere Sk3R1 32 29 Pseudomonas fluorescens group Pseudomonas mandelii strain NBRC 103147 98 SPG Rhizosphere SPG2R5 31 30 Bacillus cereus group Bacillus toyonensis strain BCT-7112 100 Sutherland Rhizosphere Su2R10 30 31 Pseudomonas fluorescens group Pseudomonas mandelii strain NBRC 103147 99 SPG Rhizosphere SPG1R4 29 32 Burkholderiaceae family Burkholderia graminis strain C4D1M 99 Elrose Rhizosphere EL3R4 28 33 Bacillus aryabhattai Bacillus aryabhattai strain B8W22 100 Contd…

Rank of Siderophore† Origin Isolate location Isolate siderophore Group Closest NCBI‡ relative Similarity(%) (µg·mL-1) production Sutherland Rhizosphere Su2R24 27 34 Pseudomonadaceae family Pseudomonas brassicacearum NFM421 99 SPG Rhizosphere SPG1R9 27 35 Burkholderiaceae family Burkholderia caledonica strain LMG 19076 99 Sutherland Rhizosphere Su2R21 26 36 Pseudomonadaceae family Pseudomonas brassicacearum NFM421 100 Skarsguard Rhizosphere Sk2R3 26 37 Pseudomonas fluorescens group Pseudomonas mandelii strain NBRC 103147 99 Skarsguard Rhizosphere Sk3R3 26 38 Pseudomonas fluorescens group Pseudomonas mandelii strain NBRC 103147 99 Skarsguard Rhizosphere Sk2R4 25 39 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 99 Skarsguard Rhizosphere Sk3R4 25 40 Pseudomonas fluorescens group Pseudomonas mandelii strain NBRC 103147 99 Elrose Rhizosphere EL3R3 25 41 Enterobacteriaceae family Leclercia adecarboxylata strain CIP 82.92 100 Elrose Endorhizosphere El3E10 25 42 Pseudomonas fluorescens group Pseudomonas poae RE*1-1-14 strain RE*1-1-14 99 Elrose Endorhizosphere El3E18 24 45 Rhizobium/Agrobacterium group Rhizobium nepotum strain 39/7 99 SPG Rhizosphere SPG1R5 24 46 Burkholderiaceae family Burkholderia caledonica strain LMG 19076 99 Laird Rhizosphere La3R9 23 47 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 100 Cabri Endorhizosphere Ca3E10 23 48 Rhizobium/Agrobacterium group Rhizobium nepotum strain 39/7 99 Laird Rhizosphere La2R15 23 49 Xanthomonadaceae Stenotrophomonas maltophilia R551-3 strain R551-3 91 SPG Rhizosphere SPG2R3 22 51 Rhizobium/Agrobacterium group Agrobacterium tumefaciens strain NCPPB2437 99 Limerick Endorhizosphere Li3E20 21 52 Pseudomonadaceae family Pseudomonas brassicacearum NFM421 100 Scott Rhizosphere Sc2R4 21 53 Burkholderiaceae family Burkholderia caledonica strain LMG 19076 99 Skarsguard Endorhizosphere Sk2E12 21 54 Pseudomonas fluorescens group Pseudomonas mandelii strain NBRC 103147 99 SPG Rhizosphere SPG3R10 20 56 Oxalobacteraceae Herbaspirillum huttiense strain NBRC 102521 99 34 Elrose Rhizosphere El1R7 20 57 Pseudomonas fluorescens group Pseudomonas poae RE*1-1-14 strain RE*1-1-14 100 Sutherland Rhizosphere Su2R22 19 61 Pseudomonas fluorescens group Pseudomonas poae RE*1-1-14 strain RE*1-1-14 99 Elrose Rhizosphere El2R9 19 62 Burkholderiaceae family Burkholderia terricola strain LMG 20594 99 Elrose Endorhizosphere El3E11 18 64 Rhizobium/Agrobacterium group Rhizobium nepotum strain 39/7 97 Laird Rhizosphere La3R7 18 65 Burkholderia cepacia complex Burkholderia pyrrocinia strain LMG 14191 99 Elrose Rhizosphere EL3R9 18 66 Pseudomonadaceae family Pseudomonas koreensis strain Ps 9-14 100 Sutherland Rhizosphere Su2R20 18 67 Pseudomonas fluorescens group Pseudomonas poae RE*1-1-14 strain RE*1-1-14 99 Sutherland Rhizosphere Su2R12 18 68 Pseudomonas fluorescens group Pseudomonas mandelii strain NBRC 103147 99 SPG Endorhizosphere SPG3E3 17 71 Rhizobium/Agrobacterium group Rhizobium nepotum strain 39/7 98 Laird Rhizosphere La2R13 17 72 Pseudomonas fluorescens group Pseudomonas poae RE*1-1-14 strain RE*1-1-14 99 Limerick Rhizosphere Li3R13 17 75 Enterobacteriaceae family Pantoea vagans C9-1 strain C9-1 99 Limerick Rhizosphere Li2R10 17 76 Pseudomonas fluorescens group Pseudomonas mandelii strain NBRC 103147 99 Laird Rhizosphere La3R8 15 84 Enterobacteriaceae family Pantoea vagans C9-1 strain C9-1 98 Laird Endorhizosphere La3E14 15 86 Rhizobium/Agrobacterium group Agrobacterium tumefaciens strain NCPPB2437 99 Laird Endorhizosphere La3E12 13 97 Rhizobium/Agrobacterium group Agrobacterium tumefaciens strain NCPPB2437 99 Elrose Rhizosphere El1R6 13 99 Burkholderiaceae family Pandoraea pnomenusa 3kgm 99 † Siderophore concentration on a Fe-free media (unreplicated) ‡ NCBI denotes National Center for Biotechnology Information

3.6 Discussion

Iron (Fe) is a very important essential nutrient in the human diet, and the requirement for Fe

can be fulfilled by consuming legumes such as lentils. Levels of Fe for Saskatchewan grown lentil, as measured by Thavarajah et al. (2009), ranged between 43 to 92 mg kg-1 and these

authors concluded that lentil is a strong candidate crops for Fe bio-fortification. In our study,

only plant shoots were assessed for Fe concentration and results indicated Fe levels ranging

between 38 to 87 mg·kg-1. However, for other crops, Bender et al. (2015) reported that nutrient content including Fe in seed of soybean (Glycine max L. Merr.) was significantly higher than in the plant biomass. Bender et al. (2015) suggested that plants grown to maturity typically have higher Fe content in the seed than the biomass. Lentil cultivars also appear to control seed Fe

levels. For example, DellaValle et al. (2013) reported that green lentil had higher relative bioavailable Fe when compared with red lentil. In their study, a green cultivar (i.e., CDC

Milestone) exhibited relatively high bioavailable Fe content grown when grown at different

locations in Saskatchewan and, for these reasons, cultivar CDC Milestone was selected in our

study as a model plant used to isolate putative potential bacteria for Fe bio-fortification.

It has been reported that availability of plant nutrients is controlled by soil properties

(Comerford, 2005). However, in our study, Fe content in plant biomass was not correlated with any of the measured soil physico-chemical properties, suggesting that there might be additional

factors involved in lentil Fe uptake and content. DellaValle et al. (2013) also reported that

location had a significant effect on Fe concentration. Specifically, they reported relative Fe

bioavailability in lentil grown in several locations in Saskatchewan and concluded that the

impact of location on lentil Fe content was more important than the impact of cultivar in their

study. In addition to variations among crops, plant bioavailable Fe differs from plant total Fe content, as often these values do not correlate with each other (Hurrell and Egli, 2010). In this study, only total Fe content of shoot biomass was measured. Results revealed that, Fe

concentration and uptake varied in a single lentil cultivar grown in soils collected from several

locations in Saskatchewan; thus, it is plausible that soil microbial properties may have influenced

lentil Fe uptake. In fact, many researchers have hypothesized that soil microorganisms including bacteria can significantly increase nutrient content and yield through enhancement of plant

growth and development (Crowley et al., 1992; Rana et al., 2015).

In this study, the isolated siderophore-producing bacteria (n = 491 isolates) from all soils produced significant amounts of siderophores in vitro. Moreover, the highest number of potential

siderophore-producing bacteria was isolated from lentil plants grown in the soil which had low

total Fe levels (Limerick soil); thus, low soil Fe conditions may stimulate populations of

siderophore-producing bacteria. Bacteria isolated from the soil from the SPG site produced the

highest average siderophore levels and isolate SPG2R11 isolated from SPG soil produces the

highest siderophore levels. Interestingly, lentil plants grown in SPG soil also exhibited

significantly higher total Fe contents and additionally, this soil also was the source of the greatest

number of siderophore-producing bacteria. Canbolat et al. (2006) and Faoro et al. (2010)

reported that bacteria populations and activity can be influenced by soil properties; however, in

the current study no relationship correlating to total heterotrophic bacteria, siderophore-

producing bacteria population and soil physico-chemical properties was observed (data not shown). It is important to note that the sensitivity of the SideroTec Assay® is reported as

approximately 2 µg mL-1; however, in the range of 50 to 100 µg mL-1, in particular, slight

variations in optical density may result in large apparently differences in estimated siderophore

production (Emergenbio, 2017). Importantly, factors other than siderophore production, such as microbial growth may also influence the optical density readings, thereby inflating or otherwise compromising estimates of siderophore production.

Many researchers reported and concluded that bacteria can effectively release nutrients in the soil from organic and inorganic sources that may than be available for plant uptake (Ingham et al., 1985). Available nutrient content can be increased with increasing the bacteria population and ultimately increase nutrient uptake and yield of crops grown under both controlled and field conditions (Siebner-Freibach et al., 2004). However, in the current study, total Fe content and shoot weight of lentil did not correlate with bacteria numbers as assessed by culture dependent methods thus suggesting that not all soils are able to host potential Fe solubilizing bacteria.

Several researchers have studied the influence of bacterial diversity or abundance on agricultural and natural soils, and concluded that bacterial diversity in soil has effects on soil and plant health (Abawi and Widmer, 2000; Garbeva et al., 2004). Researchers attempted to identified diversity using culture dependent and culture independent techniques and identification to the species level by using cultural independent molecular techniques including

DNA sequencing (Torsvik and Øvreås, 2002). Hynes et al. (2008) isolated several root- associated bacteria from legumes (lentil, pea and chickpea) grown in Canada and identified the isolates as Bacillaceae, Enterobacteriaceae, Microbacteriaceae, Micrococcaceae, Nocardiaceae and Pseudomonaceae. In the current study, a molecular-based DNA sequencing technique was used to identify siderophore-producing bacterial isolates (n=69) isolated from lentil roots.

Bacillaceae, Burkholderiaceae, Enterobacteriaceae, Oxalobacteraceae, Pseudomonadaceae,

Rhizobiaceae and Xanthomonadaceae were the dominant bacteria, which produced siderophores

in vitro. The identification is essential for microbial inoculant development and discard

pathogenic bacteria (Berg et al., 2005). Identification can be done using phenotypic

characterization or molecular methods. Molecular based identification methods are more

accurate then phenotypic identification and identity can be detected up to species level

(Clarridge, 2004). Studying different species might be an effective procedure to understand the

interactions among siderophore-producing bacteria species, soils and plants (Looney et al.,

2009).

3.7 Summary

Lentil is a crop that can potentially be used for bio-fortification due to ability to accumulate high levels of Fe in the seeds. However, lentil Fe contents can vary depending on the location at which lentil is grown. In this study, soil physico-chemical properties appeared to be a less effective parameter that affected Fe uptake by lentil.

Effective Fe solubilizing bacteria, including siderophore-producing bacteria, in the soil may increase Fe uptake in lentil as siderophore may increase nutrient solubility in soil solution and make it available for plant uptake. Siderophore-producing bacteria were isolated from lentil roots grown in eight different agricultural Saskatchewan soils and tested for colonization and siderophore production in lentils in a 45-day growth chamber study. Siderophore-producing rhizobacteria were found in both rhizosphere and endorhizosphere of lentil plants; however, siderophore-producing populations were always higher and more diverse in the rhizosphere when compared to the lentil endorhizosphere. Therefore, most of the high siderophore-producing bacteria were isolated from the rhizosphere. For example, in the current study, candidate isolates were assessed for siderophore production in vitro and out of 491 isolates, 13 isolates produced

more than 50 µg·mL-1 of siderophores. These isolates, along with an additional 27 isolates were

selected for further studies based on their ability to produce siderophores in vitro. These results indicate that potential siderophore-producing bacteria may represent a valuable alternative as a source for Fe acquisition in lentil. However, to understand their usefulness, further studies are needed to investigate the effect of siderophore-producing bacteria on additional lentil varieties.

Useful siderophore-producing bacteria isolated may facilitate the development of local/ native inoculants for crops.

4. EFFECT OF SIDEROPHORE-PRODUCING BACTERIA ON IRON CONTENT IN

LENTIL (Lens culinaris MEDIK, CV. CDC MILESTONE) UNDER CONTROLLED

ENVIRONMENTAL CONDITIONS

4.1 Preface

In this study, candidate rhizosphere and endorhizosphere siderophore-producing bacteria

from the previous study (Chapter 3) were screened for growth promotion and Fe nutrition of

lentil. The objective of this study was to assess the Fe uptake capacity of candidate bacteria on

Fe nutrition and growth promotion of lentil plants.

4.2 Abstract

The role of siderophore-producing bacteria isolates (n=40) in iron (Fe) nutrition of lentil

(Lens culinaris Medik., cv. CDC Milestone) was assessed in a growth chamber study. Lentil

seeds were inoculated with individual isolates, plants were grown in potted sand soil mixture and

harvested at 45 DAP when total-Fe was measured on plant biomass. Inoculation of siderophore-

producing isolates significantly increased total Fe uptake in 75% of inoculation treatments. The

highest Fe content (155 µg∙pot-1) was observed for plants inoculated with isolate El1R8, which produced siderophore amount of 30 µg∙mL-1 in vitro. Isolate El1R8 was previously isolated from

the rhizosphere of lentil plants and identified as Burkholderia terricola strain LMG 20594. Other

isolates e.g., SPG2R11 and SPG2R12, both identified as Herbaspirillum huttiense strain NBRC

10252, produced approximately same amount of siderophore (116 and 112 µg∙mL-1,

respectively) in vitro, but the effect of Fe uptake in plants was inverse as an increase/decrease

trend was observed in plants treated with these isolates. Results from this study indicated that

inoculation of siderophore-producing bacteria may increase total Fe uptake by lentil plants.

However, siderophore mediated Fe uptake in lentils did not reflect the in vitro siderophore production capacity exhibited by individual isolates.

Although siderophore-producing isolates may have the potential to increase Fe availability

and uptake by lentil plants thus reducing the usage of Fe fertilizer, more studies are needed to

elucidate the differences of lentil Fe nutrition observed in this study. Studies involving suitable

growth conditions and/or other nutrient uptake traits which help some low siderophore- producing isolates to stimulate Fe uptake of lentil plants would help to apply siderophore- producing bacteria to increase Fe contents of crops more effectively.

4.3 Introduction

The plant rhizobiome (rhizosphere and endorhizosphere) comprises a significant group of bacteria that could be beneficial for agriculture. Potential benefits include biofertilizer and/or biocontrol agents that improve plant growth and nutrient uptake (Weyens et al., 2009). Some of commonly found root associated bacteria include Arthrobacter, Acinetobacter, Azospirillum,

Burkholderia, Bacillus, Enterobacter, Flavobacterium, Micrococcus, Pseudomonas, Rhizobium,

Stenotrophomonas and many other groups (Germida et al., 1998; Kuklinsky-Sobral et al., 2004;

Berg and Hallmann, 2006).

Iron (Fe) is the fourth most abundant element in the earth’s crust. Iron can be present in soil solution as Fe2+ and Fe3+; however, formation of insoluble oxides and/or hydroxides of Fe limit the bioavailability of this nutrient and many of these compounds are not readily available for plants and microbes (Violante et al., 2010; Olaniran et al., 2013). Most of the food for human consumption derives from plant based sources; thus, plant Fe uptake is also important for human health. In fact, the World Health Organization (WHO) reported that Fe deficiency is the main cause of anaemia for approximately two billion people and has suggested that consumption of

Fe-rich vegetables and/or grains could be an effective solution to reduce Fe deficiency worldwide (Lozano et al., 2012; Say et al., 2014). Crop and soil management are the two key approaches that are commonly employed to source Fe for plant uptake. Crop management practices include application of inorganic Fe fertilizers (e.g., FeSO4), and application of synthetic and/or non-synthetic forms of chelated Fe as foliar treatments and/or in the root zone (Godsey et al., 2003; Álvarez-Fernández et al., 2005). It is reported that foliar application of chelated Fe can be more effective compared to an inorganic form of Fe (Fang et al., 2008). Soil management includes change in soil pH using inorganic salts (as NO or NH ) to increase Fe solubility, thus − + 3 4 enhancing plant Fe uptake (Zuo and Zhang, 2011). Chelated Fe fertilization is another way of soil management to increase available Fe in soil; however, use of metal chelators may have negative environmental impacts. For example, some chelators are phytotoxic and accumulate soluble metals that can increase total metal concentration. As a consequence, these high concentrations may also be phytotoxic to non-hyper accumulator plants, and thus may reduce plant growth and inhibit Fe uptake (McGrath and Zhao, 2003). Additionally, metal chelates can be leached to the groundwater, which may be toxic to soil macrofauna and microorganisms,

consequently affecting the stability and function of the soil ecosystem (Altieri and Nicholls,

2003).

The application of beneficial bacteria which are known as plant growth-promoting rhizobacteria (PGPR) represents another possibility to biofertilizing a soil. The PGPRs typically are considered environmentally friendly and are currently gaining acceptance. Plant growth- promoting rhizobacteria improve plant fitness through a variety of mechanisms, including the release of siderophores exhibiting a high affinity to chelate Fe (Neilands, 1995; Budzikiewicz,

2010). Many researchers have reported that plants were capable of using microbial siderophores as a means of enhancing Fe availability (Johnson et al., 2002; Álvarez-Fernández et al., 2005;

Shenker and Chen, 2005). Siderophore-producing bacteria can increase Fe solubility and may be useful for increasing plant Fe uptake and Fe content in seeds. In addition to enhancing siderophore production, plant-associated bacteria also may produce phytohormones in the rhizosphere, which may increase nutrient supply to the plants (Gamalero and Glick,; Ma et al.,

2011; Wahyudi et al., 2011).

Lentil has a high protein content and are rich in vitamins and essential micronutrients, including Fe. Lentil typically has a high Fe content among pulses and are consumed all over the world (DellaValle et al., 2013). Lentil also contains relatively high levels of antioxidants such as lycopene and β-carotene, which have a potential anticancer role in humans (Roy et al., 2010;

Torino et al., 2013; Faris et al., 2013).

Canada contributes to more than 67 percent of the global lentil export market and 90 percent of all exported lentils from Canada are cultivated in Saskatchewan (FAOSTAT, 2013; McVicar et al., 2017). As a result, lentil has become a popular pulse crop grown in the prairies.

Potentially, beneficial bacteria including PGPR can contribute significantly to the pulse industry.

The objective of this study was to investigate the effect of siderophore-producing bacteria on Fe

nutrition in lentil.

4.4 Materials and Methods

4.4.1 Soil sampling and properties

The soil was collected from the A horizon of a Brown Chernozem (Ardill association) (de

Freitas et al., 1993) located at Central Butte, SK, Canada. The soil was air dried and passed

through a 4-mm sieve before use. The loam soil had 27 g∙kg-1 organic matter (OM), pH 6.9, and

40, 30, 300 mg∙kg-1 of available N, P, K, respectively, and 21 g∙kg-1 total Fe (ALS Laboratory

Group, Saskatoon, Canada). The properties of the soil were determined as follows: texture (mini- pipet method); pH (1:2 soil: water suspension - Hendershot et al., 2008); OM (Walkley and

Black, 1934); N (CaCl2 extractable nitrate, NO3-N) (Houba et al., 2000); P and K (Modified

Kelowna extractable phosphate, PO4-P and K) (Qian et al., 1994); S (CaCl2 extractable sulphate,

SO4-S) (Houba et al., 2000); total Fe was measured in acid digestion using absorption

spectroscopy. The soil potted experiment was conducted in a growth chamber (16 h and 8 h day

and night cycle, with a 25°C: 20°C (day:night) of air temperature) at the University of

Saskatchewan, Saskatoon, SK, Canada. To ensure low nutrient levels, the soil was mixed with

silica sand (50:50 w·w–1), and 300 g of the mixture was placed in plastic pots and pre-incubated

at 80% field capacity for 10 d before seeding.

4.4.2 Measuring bacterial siderophore production

A total of 40 siderophore-producing isolates were selected for inoculation based on

siderophore production capabilities. Individual isolates were assessed in vitro for siderophore

production with two replicates. Quantitative measurement of siderophore production by each

candidate was assessed using a SideroTec® assay kit (Maynooth, Ireland;

http://www.emergenbio. com), according to the manufacturers’ instructions (Marques et al.,

2012). Briefly, all isolates were grown in 10 mL of iron-free minimal growth medium 9 (M9) at

28oC with constant shaking at 120 rpm. After incubation of 72 h, a 1.5 mL aliquot of liquid

culture was centrifuged at 5000 × g for 2 min and siderophore concentration was measured in the

cell-free supernatant. A 100 µL aliquot of supernatant was mixed with 100 µL of the assay

solution and after 10 min the absorbance was measured at 630 nm with a microplate reader

(SpectraMax®340PC, Molecular Devices, Sunnyvale, California, USA). A standard curve was created using known concentrations of siderophore provided in the SideroTec® assay kit. All

Isolates were tested further for the ability to enhance Fe uptake in lentil.

4.4.3 Seed inoculation and plant growth

Candidate siderophore-producing bacteria were grown in 150 mL Erlenmeyer flasks

containing 100 mL of 1/2 strength Tryptic soy agar (TSA) medium for 48 h at 28°C with

constant shaking at 120 rpm. After incubation, a 40 mL (n=2) aliquot of liquid culture was

placed in a sterile falcon tube and washed three times with sterile tap water using 3 cycle (each

cycle 3000 × g for 2 min) centrifugation. Harvested cultures were adjusted to their volume, and

this procedure yielded approximately 6.0 × 108 to 7.0 × 108 cfu mL–1. Individual cultures were

used to inoculate lentil (cv. CDC Milestone) seeds. Prior to inoculation, lentil seeds were surface

sterilized [70% (v·v–1) ethanol for 3 min, 1% (v·v–1) sodium hypochlorite for 1 min and washed

(n=5) with sterile tap water]. Surface sterilized seeds (n=6) and inoculum solution (1 mL per seed) were placed in separate planting holes in the soil at 15 mm depth. After germination two plants were kept in each pot. Pots were randomly organized in a growth chamber [16 h and 8 h day and night cycle, with a 25°C:20°C (day:night) of air temperature] to prevent edge effects and watered to 80% field capacity by weight. Modified Hoagland and Arnon (1950) solution containing N (211 µg∙mL-1), P (31 µg∙mL-1), K (236 µg∙mL-1), and S (64 µg∙mL-1) were added

(100 mL kg-1) to each pot prior to seed sowing and the solutions were mixed thoroughly

throughout the soil. No micronutrient was added to the soil during the assessment of

siderophore-producing bacteria on Fe uptake by lentil plant. Nutrient solution (30 mL) was added once a week by diluting the solution with the water used to maintain 80% field capacity.

Plants were harvested at flowering stage (ca., 45 DAP), and the oven dry weight of the shoot

biomass was recorded. Total-Fe concentration on plants was determined using a standard

H2SO4–H2O2 digestion method (Alcock, 1987).

4.4.4 Statistical analysis

The growth chamber studies were conducted in a completely randomized design. The

differences in Fe uptake of plants were analyzed using ANOVA at 5% significant level. The post

hoc analysis (Tukey’s test) was used to separate means using SAS software, version 9.4 (SAS

Institute Inc., Cary, N.C., USA).

4.5 Results

A total of 40 selected siderophore-producing bacteria comprising of 3 and 37

endorhizosphere and rhizosphere bacteria, respectively, were assessed for their ability to enhance

Fe uptake on lentil plants. The selected siderophore-producing bacteria produced siderophore in

vitro ranging from 11 to 116 µg∙mL-1 (Table 4.1).

The highest and lowest siderophore-producing isolates were SPG2R11 and SPG2R4, which produced 116 µg∙mL-1 and 11 µg∙mL-1 (Table 4.1), respectively. Interestingly, both bacteria were

isolated from lentil rhizosphere soil grown in the SPG soil. According to the NCBI database

results, Herbaspirillum huttiense strain NBRC 102521 was the closest relative of SPG2R11 and

Burkholderia pyrrocinia strain LMG 14191 was the closest relative of SPG2R4, with 99 percent

gene sequence similarity. Most of the siderophore-producing bacteria used in this study were

identified as Burkholderiaceae (n=18) and Pseudomonas (n=13) followed by Herbasprillum

(n=3), Bacillus (n=3), Rhizobium (n=2) and Pantoea (n=1) (Table 4.1).

Significant statistical differences were observed in total Fe uptake in lentil plants inoculated

with siderophore-producing bacteria (Table 4.2). The highest Fe content was observed for plants

inoculated with strain El1R8 (155 µg∙pot-1) and the lowest Fe content was measured in plants

inoculated with strain Su2R5 (30 µg∙pot-1). Strain El1R8 was isolated from the rhizosphere of

lentil grown in a soil collected from the Elrose location and identified as Burkholderia terricola

strain LMG 20594, with 100 percent gene sequence similarity. Strain Su2R5 isolated from the lentil rhizosphere grown in a soil from Sutherland was identified as Pseudomonas mandelii strain

NBRC 103147, with 99 percent gene sequence similarity. The amount of siderophore produced

by El1R8 and Su2R5 were 30 µg∙mL-1 and 50 µg∙mL-1 respectively. In the current study, no

significant correlation of siderophore concentration (produced in vitro) with total Fe uptake was detected (Appendix A.3) and not all siderophore-producing bacteria elicited the same response in terms of enhancing Fe uptake by lentil plants. For example, total Fe uptake in plants inoculated with the highest and lowest siderophore-producing isolates SPG2R11 and SPG2R4 was 111

µg∙pot-1 and 50 µg∙pot-1, respectively. Moreover, approximately 75% of the inoculated isolates exhibited a significant increase in total Fe uptake in the plants compared to the uninoculated control. In fact, in some cases bacterial inoculation resulted in conflicting effects (i.e., both increases and decreases in total Fe up-take), although they belonged to the same species and produced approximately the same amounts of siderophore in vitro. For example, isolates

SPG2R11 and SPG2R12 were identified as Herbaspirillum huttiense strain NBRC 102521 and produced 116 µg∙mL-1 and 112 µg∙mL-1 siderophore in vitro, respectively, but, the total Fe uptakes of the inoculated plants were 111 µg∙pot-1 and 33 µg∙pot-1, respectively.

For the next study, isolates were selected based on: (i) their effect of Fe nutrition to the plant; (ii) NCBI results and (iii) isolates exhibiting highest Fe uptake among same group.

Table 4.1 Final screening of selected (n=40) siderophore-producing candidates for siderophore production (µg·mL-1) ± SD in vitro.

Rank of Siderophore† Origin Isolate location Isolate siderophore Closest NCBI‡ relative Similarity(%) (µg·mL-1) production SPG Rhizosphere SPG2R11 116±4 1 Herbaspirillum huttiense strain NBRC 102521 99 SPG Rhizosphere SPG2R12 112±2 2 Herbaspirillum huttiense strain NBRC 102521 100 SPG Rhizosphere SPG3R9 107±3 3 Herbaspirillum huttiense strain NBRC 102521 99 Scott Rhizosphere Sc2R3 80±11 4 Burkholderia pyrrocinia strain LMG 14191 100 Laird Rhizosphere La3R8 78±7 5 Pantoea vagans C9-1 strain C9-1 98 Scott Rhizosphere Sc2R1 77±8 6 Burkholderia pyrrocinia strain LMG 14191 99 Limerick Endorhizosphere Li3E21 65±12 7 Pseudomonas brassicacearum NFM421 100 Scott Rhizosphere Sc2R2 62±14 8 Burkholderia pyrrocinia strain LMG 14191 99 Sutherland Rhizosphere Su2R5 50±8 9 Pseudomonas mandelii strain NBRC 103147 99

49 Scott Rhizosphere Sc2R11 49±4 10 Burkholderia pyrrocinia strain LMG 14191 99 Laird Endorhizosphere La3E13 46±2 11 Pseudomonas mandelii strain NBRC 103147 99 Scott Rhizosphere Sc2R10 46±4 12 Burkholderia pyrrocinia strain LMG 14191 100 Cabri Rhizosphere Ca2R1 45±1 13 Pseudomonas brassicacearum NFM421 99 Elrose Endorhizosphere El3E13 45±1 14 Rhizobium nepotum strain 39/7 100 Elrose Rhizosphere El1R5 43±5 15 Bacillus aryabhattai strain B8W22 97 Laird Rhizosphere La3R7 42±1 16 Burkholderia pyrrocinia strain LMG 14191 99 SPG Rhizosphere SPG2R9 41±8 17 Rhizobium naphthalenivorans strain TSY03b 100 Skarsguard Rhizosphere Sk2R4 39±9 18 Burkholderia pyrrocinia strain LMG 14191 99 Scott Rhizosphere Sc2R12 38±11 19 Burkholderia pyrrocinia strain LMG 14191 99 SPG Rhizosphere SPG1R1 37±11 20 Burkholderia pyrrocinia strain LMG 14191 99 Contd…

Siderophore† Rank of Origin Isolate location Isolate siderophore Closest NCBI‡ relative Similarity(%) (µg·mL-1) production Sutherland Rhizosphere Su2R21 33±5 21 Pseudomonas brassicacearum NFM421 100 Laird Rhizosphere La3R3 32±3 22 Burkholderia pyrrocinia strain LMG 14191 99 SPG Rhizosphere SPG1R2 31±0 23 Burkholderia pyrrocinia strain LMG 14191 100 SPG Rhizosphere SPG2R5 30±10 24 Bacillus toyonensis strain BCT-7112 100 Elrose Rhizosphere El1R8 30±3 25 Burkholderia terricola strain LMG 20594 100 Laird Rhizosphere La3R2 30±3 26 Burkholderia pyrrocinia strain LMG 14191 99 Sutherland Rhizosphere Su2R24 28±8 27 Pseudomonas brassicacearum NFM421 99 SPG Rhizosphere SPG1R3 28±8 28 Burkholderia pyrrocinia strain LMG 14191 100 Laird Rhizosphere La3E4 28±4 29 Bacillus aryabhattai strain B8W22 100 Skarsguard Rhizosphere Sk3R2 27±8 30 Pseudomonas mandelii strain NBRC 103147 99 Skarsguard Rhizosphere Sk3R3 26±1 31 Pseudomonas mandelii strain NBRC 103147 99 50 Limerick Endorhizosphere Li3E19 26±4 32 Pseudomonas brassicacearum NFM421 100 SPG Rhizosphere SPG1R9 25±3 33 Burkholderia caledonica strain LMG 19076 99 Skarsguard Rhizosphere Sk2R3 25±2 34 Pseudomonas mandelii strain NBRC 103147 99 Skarsguard Rhizosphere Sk3R4 25±1 35 Pseudomonas mandelii strain NBRC 103147 99 Skarsguard Rhizosphere Sk3R1 22±3 36 Pseudomonas mandelii strain NBRC 103147 98 Sutherland Rhizosphere Su2R10 21±2 37 Pseudomonas mandelii strain NBRC 103147 99 SPG Rhizosphere SPG2R6 20±4 38 Burkholderia pyrrocinia strain LMG 14191 100 SPG Rhizosphere SPG1R4 20±4 39 Burkholderia graminis strain C4D1M 99 SPG Rhizosphere SPG2R4 11±0 40 Burkholderia pyrrocinia strain LMG 14191 99 † Siderophore concentration on a Fe-free media (rep=2) ‡ NCBI denotes National Center for Biotechnology Information § SD denotes standard deviation

Table 4.2 Effect of siderophore-producing bacteria inoculation on Fe uptake in lentil cv. CDC Milestone. Adapted from Table 4.1.

Rank of Siderophore† Origin Isolate location Isolate siderophore Fe (µg·pot-1)¶ (µg·mL-1) production

Elrose Rhizosphere El1R8 30 25 155 a Limerick Endorhizosphere Li3E19 26 32 137 a Scott Rhizosphere Sc2R1 77 6 128 a Laird Rhizosphere La3R2 30 26 136 a Scott Rhizosphere Sc2R12 38 19 119 a Skarsguard Rhizosphere Sk3R2 27 30 115 ab SPG Rhizosphere SPG1R3 28 28 115 ab SPG Rhizosphere SPG2R11 116 1 111 ab Laird Rhizosphere La3R7 42 16 106 ab Skarsguard Rhizosphere Sk3R3 26 31 105 ab SPG Rhizosphere SPG1R1 37 20 102 ab SPG Rhizosphere SPG3R9 107 3 102 ab Skarsguard Rhizosphere Sk3R4 25 35 101 ab Laird Rhizosphere La3R3 32 22 99 ab Elrose Rhizosphere El1R5 43 15 94 b Laird Endorhizosphere La3E13 46 11 91 b Scott Rhizosphere Sc2R11 49 10 89 b SPG Rhizosphere SPG2R9 41 17 87 b Sutherland Rhizosphere Su2R21 33 21 84 bc Scott Rhizosphere Sc2R2 62 8 84 bc SPG Rhizosphere SPG1R2 31 23 83 bc Scott Rhizosphere Sc2R10 46 12 80 bc SPG Rhizosphere SPG2R5 30 24 78 bc Skarsguard Rhizosphere Sk2R3 25 34 76 bc

Rank of Siderophore† Origin Isolate location Isolate siderophore Fe (µg·pot-1)¶ (µg·mL-1) production

SPG Rhizosphere SPG1R9 25 33 76 bc SPG Rhizosphere SPG1R4 20 39 71 bcd SPG Rhizosphere SPG2R6 20 38 69 bcd Laird Rhizosphere La3R8 78 5 65 cd Skarsguard Rhizosphere Sk3R1 22 36 65 cd Sutherland Rhizosphere Su2R10 21 37 62 cd Elrose Endorhizosphere El3E13 45 14 57 de Sutherland Rhizosphere Su2R24 28 27 56 de Cabri Rhizosphere Ca2R1 45 13 55 de Limerick Endorhizosphere Li3E21 65 7 53 e SPG Rhizosphere SPG2R4 11 40 50 e Control 48 e Skarsguard Rhizosphere Sk2R4 39 18 47 e Laird Rhizosphere La3E4 28 29 46 e Scott Rhizosphere Sc2R3 80 4 37 f SPG Rhizosphere SPG2R12 112 2 33 f Sutherland Rhizosphere Su2R5 50 9 30 f † Siderophore concentration on a Fe-free media (rep=2) ‡ NCBI denotes National Center for Biotechnology Information ♯ Isolates in bold were selected for next study § Different letters in the same column represents significant differences at the significance level of 5% ¶ Each pot contained two plants.

4.6 Discussion

Root associated siderophore-producing bacteria may play a significant role in plant growth

through various mechanisms. Potentially, they can serve as a biofertilizer by enhancing available

nutrients including Fe, or they may act against plant pathogens to protect crop, thus offering an

excellent alternative to improve plant growth as compared to inorganic fertilizer amendments.

For example, Bar-Ness et al., (1991, 1992) reported that a siderophore-producing Pseudomonad

significantly increased Fe in oat and sorghum. Other researchers reported that siderophore- producing Pseudomonad pseudoalcaligenes significantly increased germination, root, shoot length and dry weight of Pigeon pea (Cajanus cajan), compared with the untreated control (Khan

et al., 2006; Gamit and Tank, 2014). In the current study, siderophore production was first

detected in candidate bacteria using a SideroTec® assay kit. The method uses colorimetry to

detect siderophore production (e.g., a blue color assay solution that changes to orange indicating

siderophore production). Others have similarly isolated siderophore-producing bacteria associated with various crops. For example, (Ahmad et al., 2006) used various crops to isolate 72

rhizosphere bacteria of which only eight isolates were able to produce siderophore in vitro.

In the current study, siderophore-producing isolates increased Fe content in lentil, but the degree to which the Fe content was increased did not depend on the in vitro siderophore production levels. For example, isolate SPG2R11 produced the highest siderophore in vitro, but

the response of lentil to Fe-nutrition was not the highest. A possible explanation may be related

to the inability of the isolate to produce high levels of siderophore in the soil. It has been

reported that siderophore production can be dependent on growth medium (Sharma and Johri,

2003a). Sharma and Johri (2003a) assessed siderophore production by three different

Pseudomonas spp. using four distinct growth media. They observed significant differences in siderophore production for each of the medium assessed. Similarly, Gamit and Tank (2014) reported that siderophore production could be achieved on CAS medium but was not detected when bacteria were grown in a nutrient broth medium. (Chabot et al., 1996) reported different growth promotion responses to two Rhizobium leguminosarum strains on maize and lettuce, indicating that the level of plant growth response may vary by host crop. Similarly, in the current study, SPG2R11 and SPG2R12, which both were identified as Herbaspirillum huttiense, exhibited different effects in terms of the levels of siderophore-produced in vitro, and the subsequent growth responses to these organisms when assessed in a soil-based pot experiment.

Apparently subtle differences in these organisms, likely at the biovar level, influenced their ability to enhance Fe uptake in lentil.

The success of inoculation using putative plant-growth-promoting bacteria on plant growth parameters is often restricted by several soil factors, including the presence of competing indigenous (native) bacteria. In the current study, it is possible that isolate SPG2R11 competed with the native community and, as a result, strain SPG2R11 was unable to exhibit its optimal siderophore production, previously demonstrated in vitro. Other isolates (e.g., El1R8 had a good response (highest Fe uptake to lentil), but it did not produce the highest amount of siderophore in vitro, thus suggesting that strain El1R8 may have additional and/or indirect mechanisms to enhance plant growth along with siderophore production. For example, the enhanced effect of Fe uptake observed may be related to extensive development of roots and root hairs, which increases the root surface area thus facilitating the plant to access a larger area of soil. A larger root surface increases the chance of the plant to improve micronutrients uptake (Bassirirad, 2000;

Wang et al., 2006). Beneficial microorganisms also have the potential to affect plant organs and

influence nutrient uptake. For example, results of an anatomical study by Abou-Taleb et al.

(2011) demonstrates that the development of xylem and phloem in inoculated soybean plants may have facilitated the transport of micronutrients as compared to the uninoculated plant.

Additionally, other researchers also have reported that some siderophore-producing bacteria can also decrease the pH in soil (Orhan et al., 2006), and other media (Alikhani et al., 2006) by

producing organic acids. In the current study, strain El1R8 may have been able to alter the

rhizosphere pH, which increases Fe solubility in the rhizosphere, thus promoting the increased

Fe uptake observed. This possible explanation warrants further investigation.

4.7 Summary

In the current study, 40 siderophore-producing candidate bacteria that produced varying amounts of siderophore in vitro were tested for their ability to increase Fe uptake in lentil.

Bacterial strains tested included Burkholderiaceae, Pseudomonas, Herbasprillum, Bacillus,

Rhizobium and Pantoea. Lentil seeds were inoculated with 48 hr-old cells and plants were harvested 45 d after planting. Significant increases in Fe uptake were observed in some inoculated plants. For example, the highest total Fe uptake (155 µg pot-1) was exhibited in plants

inoculated with isolate El1R8 (Burkholderia terricola). These results indicate that siderophore-

producing bacteria can increase Fe uptake in lentil. However, variable Fe uptake responses to

inoculation with siderophore-producing bacteria suggests that the use of an in vitro siderophore

production assay may not accurately identify organisms that are likely to succeed as potential

inoculants. In fact, strains SPG2R11 and SPG2R12 belong to Herbaspirillum huttiense and

produced almost the same amount of siderophore in vitro. However, their ability to enhance Fe

uptake differed when used to inoculate lentil. Thus, to better understand the effect on host plant,

it is important to assess siderophore production under non-sterile soil conditions. Additionally, in order to develop sustainable inoculant for lentils, bacteria that enhance Fe uptake in a single lentil cultivar will need to be tested further in several cultivars. Such studies will help to increase plant Fe content and ultimately increase the quality and value of Canadian lentils in the world lentil market.

5. EFFECT OF SIDEROPHORE-PRODUCING BACTERIA ON IRON CONTENT IN

DIFFERENT LENTIL AND PEA CULTIVARS

5.1 Preface

In this study, candidate siderophore-producing bacteria that exhibited superior Fe uptake in

Chapter 4 were assessed for Fe-nutrition of selected lentil and pea cultivars. The objectives of

this study were to assess the potential of siderophore-producing bacteria inoculation to increase

Fe in different lentil and pea cultivars. Moreover, potential interactions between siderophore- producing bacteria and different lentil and pea cultivars were assessed to determine if consequent plant growth promotion is cultivar specific.

5.2 Abstract

Phytosiderophores and siderophores play an important role in plant Fe-nutrition by chelating

Fe, rendering them available for crop uptake. The potential of phytosiderophore production by four lentil and four pea cultivars was assessed in vitro during germination on Chrome Azurol S agar plates. Results indicated that all lentil cultivars and one pea cultivar produced phytosiderophores, evidenced by the production of an orange halo on the agar plate assay. The effect of siderophore-producing bacteria on pea Fe uptake was assessed in a growth chamber study on plants grown in soil that previously had not grown any legumes. Plants were harvested at 45 DAP and Fe uptake was measured using atomic spectroscopy. Inoculation of pea with siderophore-producing bacteria resulted in total Fe uptake averaging 31 to 178 µg.pot-1 and all

inoculated plants exhibited higher total Fe uptake compared to the controls. For example, pea cultivar CDC Striker inoculated with isolate La3R2 exhibited 9-fold increase in total Fe uptake compared to untreated plants. Pea cultivars CDC Amarillo and CDC Dakota exhibited increases in total Fe uptake values as high as 60 µg.pot-1 when used to inoculate with isolate SPG2R11.

However, isolate SPG2R11was not effective as La3R2 when used to inoculate CDC Meadow

and CDC Striker. Similarly, La3R2 was not as effective as SPG2R11was when used to inoculate

CDC Amarillo and CDC Dakota.

Results of this study indicate that lentil and some pea cultivars are capable of

phytosiderophore production in vitro and that some pea cultivars can respond to inoculation with

siderophore-producing bacteria for total Fe uptake. However, Fe uptake by pea cultivars might

be dependent on siderophore-producing bacterial isolates for enhanced Fe nutrition and are most

responsive when inoculated with a suitable isolate.

5.3 Introduction

The World Health Organization reported that the micronutrient deficiency such as Fe

deficiency is a major problem to the human health worldwide (Milman, 2011; Miller, 2013). As

a result, billions of people are potentially subjected to Fe deficiency (Camaschella, 2015).

Although food fortification or supplementation efforts have been successful to reduce Fe

deficiency in some countries, the overall result of these strategies remain inadequate in

developing countries. Biofortification is the process of increasing nutrient content in crops

during crop growth, which offers a sustainable solution to reduce malnutrition worldwide (Bouis

and Welch, 2010). The concept of biofortification is currently gaining importance not only for

increasing crop production but also for understanding micronutrient solubilization and uptake

potential by the crop plant itself. Biofortification can be accomplished by the addition of

inorganic fertilizers, genetic engineering and/or traditional breeding to increase micronutrient

concentrations in the edible parts of crops (Xu et al., 2011; Carvalho and Vasconcelos, 2013).

However, application of inorganic fertilizers can be inefficient in some soils because fertilizers can be adsorbed on soil particles and/or be converted to insoluble forms. Additionally, repeated application of fertilizers can be costly and cause environmental pollution (Rengel et al., 1999).

Iron fertilization is very complex, because Fe can easily become insoluble (Shenker and

Chen, 2005). Synthetic chelates such as EDTA (ethylenediamine tetraacetic acid) and EDDHA

[ethylenediamine di (o-hydroxyphenylacetic) acid] are currently available commercially and are commonly used in agriculture (Altieri and Nicholls, 2003). However, both EDTA and EDDHA are not easily biodegradable in the soil and can become an environmental pollutant (Álvarez-

Fernández et al., 2005). Moreover, EDTA is not easily removeable from environmental materials through wastewater treatment (Kim and Ong, 1999). In fact, the main threat is the accumulation of both EDTA and EDDHA in the ground water (Huang et al., 1997). Although high concentrations of EDTA is nontoxic to humans, it can form chelated compounds with elements such as heavy metals rendering these materials both toxic and water soluble, thus resulting in contaminated water unacceptable for human consumption (Huang et al., 1997).

Foliar applications of soluble Fe are commonly used to increase yields of crops grown in Fe- deficient soils. Although this approach increases Fe final concentrations in the crop, frequent foliar applications are needed thus reducing the feasibility of this method for large-scale

agriculture operations. For these reasons, it is very important to find alternatives to increase

available Fe for plant uptake, specifically for those plants which are sensitive to Fe deficiency

(Fang et al., 2008; Aciksoz et al., 2011).

Plants use two approaches for Fe uptake (Marschner and Römheld, 1994). Approach (I)

includes reducing the pH of the rhizosphere and reduction of Fe3+ ions using membrane-bound

Fe3+ reductase followed by consequent Fe2+ uptake by root cells. Approach (II) refers to the

plants which produces low molecular weight chelating compounds called phytosiderophores.

Phytosiderophores can solubilize and chelate Fe which is transported into root cells through

membrane proteins (Crowley et al., 1992; Zuo and Zhang, 2009; Dotaniya et al., 2013; Bocchini

et al., 2015). However, approaches I and II alone may not be sufficiently effective enough to

supply adequate levels of Fe needed for plants to grow, particularly those crops grown in

calcareous and alkaline soils (Altomare and Tringovska, 2011).

Some rhizosphere microorganisms exert direct and/or indirect mechanisms that facilitate

plant growth including increases in metal solubility in the soil rhizosphere for plant uptake

(Mishra et al., 2011). A direct plant growth promotion mechanism refers to increase plant

nutrient accessibility or synthesis of phytohormone-like compounds which reduces soil pH and increase nutrient availability including Fe (Dakora and Phillips, 2002). Furthermore, additional plant growth and development regulating phytohormones including auxins, cytokinins, and gibberellins can also be synthesized by microbes (Costacurta and Vanderleyden, 1995). Rana et al. (2015) reported that inoculation of siderophore-producing bacteria in rice and wheat increased root and shoot biomass and Fe accumulation in grain. Ma et al. (2009) also reported similar results in mustard plants inoculated with PGPB Achromobacter xylosoxidans AX10.

Indirect mechanisms are mostly referred to plant protection against pathogens as some bacteria act as biocontrol agents (Glick, 2012). Some mode-of-actions utilized by rhizobacteria to protect crops include: (i) Production of antifungal metabolites and antibiotics; (ii) Induced systematic resistance; (iii) Competition for nutrients; and, (iv) Niche exclusion. Yu et al. (2011) successfully reduce Fusarium wilt of pepper using Bacillus subtilis CAS15 by induced systemic resistance.

It is well known that siderophores can chelate Fe from insoluble sources in the soil and make it available for plant uptake (Glick, 2012); however, not all plants use siderophores. For example, certain plant species including barley, oat, tomato, sorghum, rice, and wheat exhibit different abilities to use siderophores (Cline et al., 1984; Crowley and Kraemer, 2007; Carvalhais et al.,

2013). Other plant species are able to select specific siderophore-producing bacteria to release siderophores into their rhizosphere and both organisms (host plant and bacteria) may use these bacterial siderophores (Crowley et al., 1988; Rengel et al., 1999).

Pulses are a part of the regular diet of many vegetarians as well as of people from some developing countries and pulse consumption is increasing worldwide at very high rate compared to other crops. Annual world lentil and field pea production are now approximately 5.2 million tons and 11.5 million tons, respectively, of which 2.2 million tons (22%) of lentil and 4 million tons (30%) of field pea are grown in Canada, primarily in Saskatchewan (FAOSTAT, 2013).

Currently, the lentil production in Canada is gradually increasing and more than 90% of

Canadian pulses are exported to more than 100 countries in South Asia, Middle East and Europe

(McVicar et al., 2017).

The objectives of this study were to assess the phytosiderophore production by different

lentil and pea cultivars and identify whether siderophore-producing bacteria isolated from

Saskatchewan soils are cultivar specific in terms of enhancing Fe nutrition to the pea plants.

5.4 Materials and methods

5.4.1 Soil preparation and seed collection

Lentil and pea cultivars were selected based on marketability in Bangladesh (e.g., commonly marketed in Bangladesh). Thus, four lentil cultivars (CDC Maxim, CDC Dazil, CDC 3646-4 and

CDC Milestone) and four pea cultivars (CDC Meadow, CDC Amarillo, CDC Striker and CDC

Dakota) were used in this study. A loamy Brown Chernozem of the Ardill association soil was used for this experiment. The soil was collected from the top 15 cm located at Central Butte, SK,

Canada (Qian and Schoenau, 2005). The soil was air dried and passed through a 4-mm sieve before use. The soil had 27 g∙kg-1 organic matter, pH 6.9, and 40, 30, 300 mg∙kg-1 of available N,

P, K, respectively, and 21 g∙kg-1 total Fe (ALS Laboratory Group, Saskatoon, Canada). The

properties of the soil were determined as follows: texture (mini-pipet method); pH (1:2 soil:

water suspension - Hendershot et al., 2008); organic matter (Walkley and Black, 1934); N (CaCl2

extractable nitrate, NO3-N - Houba et al., 2000); P and K (Modified Kelowna extractable

phosphate, PO4-P and K - Qian et al., 1994); S (CaCl2 extractable sulphate, SO4-S - Houba et al.,

2000); total-Fe was measured in acid digestion using absorption spectroscopy (Alcock, 1987).

5.4.2 Phytosiderophore production assay

Lentil and pea cultivars were tested for phytosiderophore production using a solid agar plate assay (Alexander and Zuberer, 1993; Reichman and Parker, 2007). Lentil and pea seeds were surface sterilized in ethanol and sodium hypochlorite, and four surface sterilized seeds of each cultivar were placed on a Chrome Azurol S (CAS) plate (Alexander and Zuberer, 1991). Each plate was replicated three times. Plates were incubated in the dark at 25°C:20°C (day:night) during germination and exposed to light for 18h:6h day:night cycle after germination.

Phytosiderophore production was assessed for up to 7 d by measuring the size of the halo. Two isolates (Li3E20 and Ca2E4) that produced different distinct halos in vitro (Chapter 3, Table

A.2) and produced 21 µg∙mL-1 and 6.5 µg∙mL-1 siderophore, respectively, were used for

comparison of halo development during the assay. Isolates Li3E20 and Ca2E4 were transferred

on CAS plate and incubated at the same time and under the same experimental conditions.

5.4.3 Growth chamber assessment of growth enhancement by siderophore-producing bacteria

A pot experiment was conducted in a growth chamber [16 h and 8 h day and night cycle,

with a 25°C: 20°C (day:night) air temperature] at the University of Saskatchewan, Saskatoon,

SK, Canada. Plastic pots containing 1.0 kg of soil as described previously in (Section 5.4.1) were

pre-incubated at 80% field capacity for 10 d before seeding. Modified Hoagland and Arnon

(1950) solution containing N (211 µg∙mL-1), P (31 µg∙mL-1), K (236 µg∙mL-1), and S (64 µg∙mL-

1) was added (100 mL kg-1) to each pot prior to seed sowing and the solutions were mixed

thoroughly throughout the soil. Seeds were inoculated (n=4) with siderophore-producing bacteria

selected as described in Chapter 4, including isolates El1R8 (Burkholderia terricola strain LMG

20594), Li3E19 (Pseudomonas brassicacearum NFM421), La3R2 (Burkholderia pyrrocinia

strain LMG 14191), Sk3R2 (Pseudomonas mandelii strain NBRC 103147) and SPG2R11

(Herbaspirillum huttiense strain NBRC 102521). Surface sterilized seeds (n=6) and each bacterium culture were placed in each pot at 1.5 cm depth. Plants were thinned to two plants per pot after germination and emergence. Nutrient solution (100 mL pot-1) was added once a week by diluting the solution with the irrigation water. Water was added in alternate days and the pots were maintained at 80% field capacity. Plants were harvested 45 days after planting (DAP) and shoot fresh and dry biomass were recorded. Plants were dried at 65°C for 72 h in a ventilated oven, ground in a mortar and pestle and processed for nutrient content including total-N, -P and -

Fe in H2SO4–H2O2 digestions, as previously described (Chapter 4).

5.4.4 Statistical analysis

The growth chamber studies were conducted using a completely randomized design.

Statistical differences in the effect of siderophore-producing isolates were analyzed using

ANOVA at a 0.1% significant level. The post hoc analysis (Tukey’s test) was used to separate means using SAS software, version 9.4 (SAS Institute Inc., Cary, N.C., USA). Principal

Components Analysis (PCA) were made using JMP software (JMP® Pro, version 12, SAS

Institute Inc., Cary, N.C., USA) to demonstrate the overall effect of siderophore-producing bacteria inoculation on biomass, nutrient concentration and total nutrient uptake by pea plants.

5.5 Results

5.5.1 Phytosiderophore production assay

Four lentil and four pea cultivars were tested on CAS medium to study phytosiderophore

production. All lentil cultivars produced orange halos in the CAS medium during seed

germination, which is an indication of phytosiderophore production. The sizes of the halos were

approximately the same for all lentil cultivars (Fig. 5.1a). Only one pea cultivar (CDC Dakota) produced an orange halo during the germination process. The halo surrounding the germinating pea seed was similar to those produced by lentil cultivars, except that the halo color produced by

Li3E20 (Pseudomonas brassicacearum NFM421) exhibited a more intense color, which suggests this bacterium was able to produce siderophores at a greater concentration than the lentil or pea seeds (Fig. 5.1b).

A B

Fig. 5.1a Lentil cultivars CDC Dazil, CDC Milestone, CDC 3646-4 and CDC Maxim (A) and pea cultivars CDC Striker, CDC Dakota, CDC Meadow and CDC Amarillo (B) on Chrome Azurol S medium during germination at 5 days.

Fig. 5.1b In vitro siderophore production by isolates Li3E20 and Ca2E4 on Chrome azurol S medium.

5.5.2 Growth chamber assessment of growth enhancement by siderophore-producing bacteria

Pea plants inoculated with siderophore-producing bacteria were harvested at 45 DAP and plant total Fe contents were measured. Total Fe uptake in pea ranged from 19 to 178 µg.pot-1 and

total Fe uptake were slightly higher in all inoculated treatments compared to untreated controls

(Table 5.1). However, all cultivars and isolates did not exhibit the same inoculation response. For

example, CDC Striker exhibited a very high increase in Fe uptake in all bacterial inoculant

treatments compared to other cultivars. The highest Fe uptake (178 µg.pot-1) in CDC Striker was

associated with inoculation with isolate La3R2, which exhibited an observed 9-fold increase

relative to uninoculated CDC Striker plants (Table 5.1). In addition, isolate La3R2 also exhibited

the highest Fe uptake when inoculated in cultivars CDC Meadow, although this isolate did not

produce the highest amount of siderophore in vitro, previously (Chapter 3, Table A.2). Two

cultivars (CDC Amarillo and CDC Dakota) inoculated siderophore-producing isolate SPG2R11

exhibited the highest total Fe uptake compared to their other treatments and interestingly the

same amount of total Fe uptake (60 µg.pot-1). In contrast, isolate SPG2R11 did not show similar

response as La3R2 when inoculated in CDC Meadow and CDC Striker. Furthermore, isolates

La3R2 and El1R8 produced similar amounts of siderophore (30 µg. mL-1) in vitro (Chapter 4,

Table 4.2), but the response was inverse when CDC Meadow was treated with La3R2, which exhibited 77 µg.pot-1 Fe, whereas inoculation with El1R8 exhibited 41 µg.pot-1 Fe (Table 5.1).

Table 5.1 Iron uptake in pea plants inoculated with siderophore-producing bacteria, grown in Brown (Ardill association) soil for 45 days in a growth chamber.

Rank of Total Fe uptake Siderophore† Variety Isolate Origin Isolate location siderophore Closest NCBI‡ relative Similarity(%) (µg·pot-1) (µg·mL-1) production SPG2R11 SPG Rhizosphere 60 defghi 116 1 Herbaspirillum huttiense strain NBRC 102521 99 El1R8 Elrose Rhizosphere 56 defghi 30 25 Burkholderia terricola strain LMG 20594 100 La3R2 Laird Rhizosphere 40 hijkl 30 26 Burkholderia pyrrocinia strain LMG 14191 99 SK3R2 Skarsguard Rhizosphere 37 ijklm 27 30 Pseudomonas mandelii strain NBRC 103147 99 Amarillo Li3E19 Limerick Endorhizosphere 31 klm 26 32 Pseudomonas brassicacearum NFM421 100 Control 22 lm SPG2R11 SPG Rhizosphere 60 defgh 116 1 Herbaspirillum huttiense strain NBRC 102521 99 Li3E19 Limerick Endorhizosphere 58 defghi 26 32 Pseudomonas brassicacearum NFM421 100 La3R2 Laird Rhizosphere 53 efghij 30 26 Burkholderia pyrrocinia strain LMG 14191 99 El1R8 Elrose Rhizosphere 46 ghijk 30 25 Burkholderia terricola strain LMG 20594 100 Dakota

6 8 SK3R2 Skarsguard Rhizosphere 37 hijklm 27 30 Pseudomonas mandelii strain NBRC 103147 99 Control 32 klm La3R2 Laird Rhizosphere 77 cd 30 26 Burkholderia pyrrocinia strain LMG 14191 99 Li3E19 Limerick Endorhizosphere 75 cde 26 32 Pseudomonas brassicacearum NFM421 100 SK3R2 Skarsguard Rhizosphere 72 cdef 27 30 Pseudomonas mandelii strain NBRC 103147 99 SPG2R11 SPG Rhizosphere 50 fghijk 116 1 Herbaspirillum huttiense strain NBRC 102521 99 Meadow El1R8 Elrose Rhizosphere 41 hijkl 30 25 Burkholderia terricola strain LMG 20594 100 Control 34 jklm La3R2 Laird Rhizosphere 178 a 30 26 Burkholderia pyrrocinia strain LMG 14191 99 Li3E19 Limerick Endorhizosphere 114 b 26 32 Pseudomonas brassicacearum NFM421 100 El1R8 Elrose Rhizosphere 108 b 30 25 Burkholderia terricola strain LMG 20594 100

Striker SPG2R11 SPG Rhizosphere 95 bc 116 1 Herbaspirillum huttiense strain NBRC 102521 99 SK3R2 Skarsguard Rhizosphere 67 defg 27 30 Pseudomonas mandelii strain NBRC 103147 99 Control 19 m ‡ NCBI denotes National Center for Biotechnology Information

§ Different letters in the same column indicate significant differences at α=0.05 using Tukey’s post hoc test

ANOVA results demonstrate significant variation in total Fe uptake in pea plants amongst all inoculation treatments (Table 5.2). The interaction or combine effect of variety and isolate was also significantly different for Fe uptake.

Table 5.2 Effect of siderophore-producing bacteria inoculation on total Fe uptake in different pea cultivars.

df Mean Square F value P (>F)

Cultivar 3 6721 50.87 1.80E-12 ***

Isolate 5 4788 36.24 1.76E-12 ***

Cultivar × Isolate 15 1566 11.85 2.95E-09 ***

Residuals 33 132

P values ≤ 0.001 are significant at 0.1% level (***).

All treatments exhibited an increased in nutrient content and shoot weight compared to the uninoculated controls (Table A.5). Principal component analysis revealed that the controls of all

treatments grouped together and vectors indicated the impact of inoculation on nutrient content

and shoot weight (Fig. 5.2).

Concentration

Uptake

Controls

Fig. 5.2 Principal component analysis (PCA) of nutrient concentration and uptake patterns in 45 DAP pea cultivars CDC Amarillo, CDC Dakota, CDC Meadow and CDC Striker inoculated with 5 siderophore-producing bacteria isolates. On the x axis is the percentage of variation explained by principal component 1 (57.5%); on the y axis is the percentage of variation explained by principal component 2 (30.9%).

5.6 Discussion

Plants can produce phytosiderophores in their root zone to increase Fe solubility thereby playing an important role in Fe availability and uptake from the soil (Crowley et al., 1991).

Many crops including barley, oat, rice, sorghum, tomato, wheat have been investigated previously for phytosiderophore production (Marschner et al., 1986; Briat, 2007). In the current study, all lentil cultivars and one pea cultivar (CDC Dakota) were able to produce phytosiderophore-like compounds during germination. Researchers also reported that lentil and pea can also produce polyphenols that can bind to Fe (Sandberg, 2002). Additionally, the change of the color in the CAS medium could be attributed to the presence of polyphenols and/or other organic compounds that could have been released during the germination of lentil and pea

(d’Arcy-Lameta, 1986, Dakora et. al., 2002). Published reports indicated that the release of phytosiderophores differs among specie,s and phytosiderophore production can vary in terms of crop cultivars, age of the plant, available micronutrient status of the soil and daylength (Clark et al., 1988; Cakmak et al., 1996). Daneshbakhsh et al. (2013) studied phytosiderophore in different wheat cultivars and reported that cultivars Cross and Rushan produced highest phytosiderophore levels in a hydroponic system. In the current study however, phytosiderophore production yielded similar halos by four lentil and one pea cultivars during germination. A quantitative phytosiderophore assay could represent more accurate differences on phytosiderophore production. Gries et al. (1995) reported that high amount of phytosiderophore released by barley plant at 10 DAP, whereas wheat plants exhibited the highest release at 21 DAP. Additionally, phytosiderophore release can fluctuate between cultivars of a same species at different plant age as cultivars Cross and Rushan released highest phytosiderophore at 21 and 28 DAP, respectively

(Daneshbakhsh et al., 2013). In the current study, phytosiderophore release was assessed only

during germination in vitro on CAS agar plates, thus a study that included growing plants to

maturity would have provided a better understanding of the phytosiderophore release by lentil

and pea cultivars. Moreover, phytosiderophores can be released in the plant’s rhizosphere in a

range of 4 to 6 hours daily depending on day lighting period (Marschner et al., 1987; Oburger et

al., 2014). The rate of release is also dependent on nutrient status of the growth medium such as

Fe and/or Zn as phytosiderophore release may increase with the decrease of Fe and/or Zn (Gries

et al., 1995). However, in the current study, no relation was observed on agar plates containing

low Fe content; thus, varying amount of Fe in the growth medium might help explain the effect of Fe on phytosiderophore release by the crop.

Ishimaru et al. (2006) and Daneshbakhsh et al. (2013) reported that high phytosiderophore-

producing plant species such as barley, rye and wheat can be more resistant to Fe deficiency than

low phytosiderophore-producing crops such as maize, sorghum and rice. The phytosiderophore

released by plants subsequently form Fe phyto siderophore complexes, which later are absorbed

by plants. However, the uptake rate of Fe phytosiderophore complex varies between plant

species. Romheld and Marschner (1990) reported that phytosiderophore absorption differences

between plant species could be related to root morphology. In their study, the highest

phytosiderophore uptake rates were found in plant species with fine and highly branched root

system present in rice and sorghum and lowest uptake rates were exhibited in plant species

exhibiting relatively dense root system such as maize. Additionally, phytosiderophore uptake

rates can vary among different cultivars of the same plant species. In the current study however,

the pea cultivar CDC Striker did not produce phytosiderophore when tested on CAS agar plate

during germination, but this cultivar exhibited the highest Fe uptake when compared to the

phytosiderophore-producing pea cultivar CDC Dakota when grown under the same conditions.

The plausible cause could be related to the fact that CDC Striker was producing

phytosiderophores at a different growing period and/or had additional mechanisms other than

phytosiderophore production, which helped to increase Fe uptake (Kim and Guerinot, 2007;

Kobayashi and Nishizawa, 2012). Studies involving phytosiderophore and/or phytohormone

production by pea cultivars at different plant ages may help to identify possible Fe uptake mechanisms more precisely.

The effect of siderophore producing bacteria on Fe nutrition has been studied by researchers on numerous crops (Shenker and Chen, 2005; Lemanceausoils et al., 2009; Bhattacharya, 2010).

Results reported by Rana et al. (2015) clearly demonstrated that the siderophore-producing bacteria significantly increased Fe concentration in wheat and rice grains and it was suggested that siderophores can increase Fe concentration in the root zone of the plants, which in turn enhanced root activity and ultimately improved plant Fe uptake, including seed Fe content. In the

current study, pea cultivars exhibited a great response to inoculation with siderophore-producing

bacteria for Fe uptake, which possibly could have resulted in significant increases in total Fe

uptake in the pea plant biomass. Similar results have been published by Mishra et al. (2011), who

reported a two-fold increase of lentil seed Fe concentration when inoculated with Pseudomonas

species. Similarly, De Santiago et al. (2011) reported inoculation of wheat with a siderophore-

producing strain Trichoderma asperellum resulted in a 1.5-fold enrichment in the shoot biomass

Fe concentration. Sharma et al. (2013) reported a two-fold translocation efficacy of Fe from roots

to grains when inoculated with siderophore-producing bacterial strains Pseudomonas fluorescens, Pseudomonas putida and Azospirillum lipoferum. In the current study, isolate La3R2

(Burkholderia pyrrocinia strain LMG 14191) significantly increase total Fe uptake in pea.

Studies conducted by Hynes et al. (2008) and Rana et al. (2012) with lentil, pea, canola and rice demonstrated the importance of inoculant specificity and plant species. In the current study, we observed differing responses which were pea cultivar specific; thus our findings support their observations on inoculant specificity. For example, isolates La3R2 and SPG2R11 that were used in pea were most effective in enhancing Fe uptake amongst the five siderophore-producing bacteria tested. Furthermore, isolate La3R2 effectively increased Fe uptake in cultivars CDC

Striker and CDC Meadow. Additionally, isolate SPG2R11 also effectively increased Fe uptake in

CDC Amarillo and CDC Dakota. These results indicate that siderophore-producing isolates that significantly increase Fe uptake of one cultivar may not perform similarly on other cultivars under the same plant growth conditions. These variations could be related to: (i) soil properties and/or (ii) to the genotype of the host plant or (iii) the siderophore-producing bacterial isolates.

However, in the current study, any relation between siderophore-producing isolates and the soil properties of their origin was not observed, although they were isolated from Saskatchewan soils exhibiting distinct soil properties including organic matter, pH, and available-N, -P, -K and total-

Fe contents. Additionally, the differences in the efficiency of siderophore-producing bacteria in pea might be explained by functional and genetic differences at the host-bacteria interface level

(Mori, 1999; Kobayashi and Nishizawa, 2012).

Mimmo et al. (2014) reported that the level of siderophore production in growth medium plays an important role in Fe nutrition of the host plant and bacteria, and suggested that siderophores produced by bacteria are beneficial to the host plant. Results of the current study support this hypothesis as isolate SPG2R11, which produced the highest levels of siderophore compared to other isolates, also increased Fe uptake in some pea cultivars when grown under

similar growth conditions. In contrast, a low siderophore-producing isolate such as La3R2 can also be effective in some pea cultivars; however, isolates El1R8 and La3R2, which exhibited

similar capability to produce siderophore in vitro, differed in their ability to enhance Fe uptake,

with isolate La3R2 outperforming El1R8 in terms of the plant response. Several mechanisms

have been reported to explain inconsistencies in Fe uptake in bacterial-inoculated plants. These

reports indicated that growth promotion mechanisms other than siderophore production may be

involved in Fe uptake by the host plant. For example, hormone-producing rhizobacteria can increase Fe uptake in the rhizosphere (Pii et al., 2015; Scagliola et al., 2016). Phytohormone production may be attributable to the organisms used in the current study, which in turn may have stimulated Fe uptake in pea. Several reports (Dunn et al. 2006; Hibbing et al. 2010; Dakora and Phillips 2002; Karakashev et al. 2005) have indicated that that competition with indigenous microorganisms or scarcity of suitable environmental conditions may lead to low performance of

an introduced inoculant.

Methods to clearly distinguish siderophores produced by different isolates during plant

growth are relatively scarce (Neilands, 1995; Hider and Kong, 2010) and it is difficult to draw

concrete conclusions about plant and microbe competition for siderophere in soil conditions.

However, researchers have used radioactive Fe to study siderophore-mediated Fe uptake in various crops and cultivars. Bar-Ness et al. (1991, 1992) and Yehuda et al. (1996) reported that

radioactive Fe chelated with bacterial siderophore significantly stimulated Fe uptake in barley,

maize, oat, peanut and sorghum plants. Our study identified five siderophore-producing isolates

from Saskatchewan field soils that differed in their efficiency to contribute to Fe uptake of pea

cultivars. These include isolates La3R2 and SPG2R11 that were very competitive and highly

effective for four specific pea cultivars grown in a nonsterile soil. The effectiveness of these

isolates on other crops grown in different Saskatchewan soils needs to be evaluated to establish

crop and soil specificity.

5.7 Summary

Siderophore and phytosiderophore production by bacteria and plants, respectively, can

increase plant Fe uptake. In this study, phytosiderophore production by different lentil and pea

cultivars was assessed in vitro for phytosiderophore production. Subsequently, different pea

cultivars were inoculated with siderophore-producing bacteria and the impact on Fe nutrition was

assessed. Results obtained from this study indicated that one pea and four lentil cultivars were capable of phytosiderophore and/or polyphenol production in vitro. To confirm the cause of halo production in the CAS medium during germination of lentil and pea, different polyphenol and or other organic compounds produced by lentil and pea roots needs to be investigated extensively in

the future. The inoculation of siderophore-producing bacteria increased total Fe uptake in all four pea cultivars. However, inoculation of pea with specific siderophore-producing bacteria

significantly increased total Fe uptake in specific pea cultivars thus indicating a possible plant

bacteria specificity. Additionally, total Fe uptake varied amongst cultivars, with pea cultivar

CDC Striker exhibiting the highest total Fe uptake although this cultivar did not exhibit

phytosiderophore production in vitro, suggesting that phytosiderophore and/or polyphenol

production may not be the mechanism that increases Fe uptake in pea. Further studies using

siderophore-producing bacteria as inoculants in agricultural crops would help to determine Fe

uptake potential and identify plant host and bacterium specificities.

6. SYNTHESIS AND CONCLUSIONS

Iron deficiency is a major nutritional disorder worldwide. Biofortification is a technique which aims to decrease human Fe deficiency by increasing micronutrients in staple crops. Plant growth-promoting bacteria have potential to fortify Fe content within plants through various mechanisms. The overall goal of this study was to examine the potential of siderophore- producing bacteria to stimulate Fe uptake in lentil and pea cultivars that are adapted to

Saskatchewan agriculture. Initial growth chamber and laboratory experiments were conducted to isolate siderophore-producing bacteria associated with lentil roots. The first growth chamber study (Chapter 3) was conducted using soils collected immediately after harvest from eight different Saskatchewan fields where lentil was grown. The identity of culturable siderophore- producing bacteria isolated from lentil roots varied significantly among locations. The population of siderophore-producing bacteria was less than one percent of the total culturable root associated heterotopic bacteria. When tested for siderophore production, bacteria isolated from lentil grown in SPG soil exhibited the highest average in vitro siderophore production, and lentil grown in this soil also exhibited the highest total Fe uptake, thus suggesting that siderophore production might have stimulated Fe uptake in lentil. Others have similarly hypothesized that soil microorganisms including bacteria can significantly increase crop nutrient content (Mishra et al. 2013; Rana et al. 2012). However, in this study, the variation in siderophore-producing bacteria population and Fe uptake by a single lentil cultivar grown in different soils were not correlated to the physico-chemical properties of the soil.

Isolates exhibiting higher siderophore production in vitro were identified using Sanger

sequencing method (16S rRNA gene sequencing), and were found to belong to the following

families: Bacillaceae, Burkholderiaceae, Enterobacteriaceae, Oxalobacteraceae,

Pseudomonadaceae, Rhizobiaceae and Xanthomonadaceae. This group of bacteria reportedly were also commonly found in crops including lentil, pea and chickpea (Hynes et al., 2008).

A second growth chamber study (Chapter 4) was carried out to evaluate the inoculation effect of 40 siderophore-producing bacteria, identified previously on Chapter 3, on a lentil

cultivar CDC Milestone grown in a soil collected from commercial field with no known history

of legume production. Inoculation of siderophore-producing isolates significantly increased total

Fe uptake by lentil plants in 75% of the treatments. The five isolates observed to increase Fe uptake in lentil plants (Chapter 3) were subsequently inoculated in a third growth chamber study

(Chapter 5) in which different pea cultivars were grown to assess Fe uptake on pea. The findings

from these two growth chamber studies clearly demonstrated the capability of selected

siderophore-producing isolates to stimulate Fe uptake in both lentil and pea. However, in this

study the contribution of siderophore-producing isolates to Fe nutrition in plants was not clearly related to assessments of in vitro siderophore production. For example, the highest in vitro siderophore-producing isolate SPG2R11 significantly increased Fe uptake only in some pea cultivars, but not all. This dissimilarity of in vitro siderophore production and inoculation results might be attributed to the differences in the impact of growth medium and growing conditions on siderophore-producing bacteria. Many researchers have reported that siderophore production varies among growth medium and some isolates have been observed to have more than one

growth promotion trait (Botelho and Mendonça-Hagler, 2006). The multiple growth promotion

traits include impacts on root and root hair development, xylem and phloem development, ACC

(1-aminocyclopropane-1-carboxylic acid) activity, and lowering soil pH by producing organic acids, all of which can lead some low siderophore-producing isolates to stimulate Fe uptake in plants.

There were some interactions between siderophore-producing bacteria and the host plant, indicating a level of host plant specificity. For example, isolate El1R8 resulted in the highest Fe uptake in lentil (Chapter 4) whereas inoculation of pea with La3R2 resulted in the highest Fe uptake (Chapter 5). Furthermore, the response of different pea cultivars to different inoculants varied. These results indicated a level of host plant specificity. In a similar study Hynes et al.

(2008) and Rana et al. (2015) have been reported the host-bacteria specificity for plant growth and nutrient uptake.

Plant growth response also suggested that various mechanisms including phytosiderophore production may contribute to enhanced plant growth and nutrient uptake. Phytosiderophores can chelate Fe and make it available for plant uptake. Phytosiderophore mediated Fe uptake has been reported by many researchers (Cesco et al., 2006; Ishimaru et al., 2006; Dotaniya et al., 2013).

However, in the current study, all lentil cultivars tested and one pea cultivar exhibited phytosiderophore production during an in vitro germination bioassay, but the phytosiderophore- producing pea cultivar did not have significantly greater Fe uptake compared with the other non- phytosiderophore-producing cultivars. Phytosiderophore production is known to be dependent on age of the plants, so a long growing period might help further elucidate the role of phytosiderophore production on uptake of Fe by pea and lentil.

6.1 Future perspectives

Application of siderophore producing bacteria increased total Fe uptake of lentil and pea plants, thus, inoculation of siderophore producing bacteria could be a promising and cost- effective approach to increase Fe contents in plants. In the current study, although lentil and one pea cultivar produced phytosiderophores in vitro, this did not prove to be an effective mechanism of enhancing Fe uptake, but many researchers have reported that phytosiderophore can stimulate

Fe uptake. Thus, siderophore and phytosiderophore mediated Fe uptake in plant have opened new doors for research. The siderophore and phytosiderophore production, as well as the relationship between siderophore producing bacteria and crops in different soil conditions are still unknown. Additional studies involving different plant cultivars, siderophores and phytosiderophores could help to clarify the steps that are involved in enhancing Fe uptake for all crops. Evaluation of genetic information of plants and microbes with comprehensive characterization may help to identify methods that could be used more effectively during the search for siderophores. A better knowledge of the chemical structures and functions of these compounds would lead to various applications. The differences of their functional and structural properties in relation to microbial communities and plant species need to be deeply investigated to improve our understanding of the mechanisms leading to enhanced Fe uptake. Additionally, more research is needed to find effective ways of using siderophore-producing bacteria and phytosiderophore-producing plants in agriculture to enhance human Fe nutrition.

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8.APPENDIX

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Table A.1 Shoot dry weight, Fe concentration and total uptake by lentil (CDC Milestone) grown in growth chamber in eight distinct Saskatchewan soils (Mean ± SD) at 45 days.

Dry shoot weight Fe concentration Total Fe uptake Location (g·pot-1) (µg·g-1) (µg·pot-1)

Cabri 2.99±0.04 abc 59± ab 175±22 abc Elrose 2.81±0.11 abcd 63± ab 178±27 abc Laird 3.59±0.13 a 53± ab 187±44 ab Limerick 3.10±0.21 ab 53± ab 163±19 abc Scott 2.16±0.25 bcd 50± ab 107±27 bc Skarsgard 1.83±0.66 cd 38± b 68±13 c Spg 3.87±0.13 a 71± ab 267±27 a Sutherland 1.66±0.41 d 87± a 145±2 bc

Different letters indicate significant differences for each column at α=0.05 using Tukey’s post hoc test.

Fig A.1 Growth curve (Klett units) of randomly selected siderophore-producing bacteria in iron- free minimal medium (M9) at 28°C for 7 days. Isolate rankings refer to the in vitro siderophore production rankings amongst the 491 isolates tested subsequently.

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Table A.2 In vitro siderophore production by liquid cultures of putative siderophore-producing bacteria at 72 h assessed on sideroTec® assay kit.

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Sutherland Rhizosphere Su2R21 26.26 36 Skarsguard Rhizosphere Sk2R3 26.01 37 Skarsguard Rhizosphere Sk3R3 25.98 38 Skarsguard Rhizosphere Sk2R4 25.36 39 Skarsguard Rhizosphere Sk3R4 25.28 40 Elrose Rhizosphere EL3R3 24.98 41 Elrose Endorhizosphere El3E10 24.84 42 Elrose Endorhizosphere El3E19 24.72 43 Elrose Endorhizosphere El3E17 24.02 44 Elrose Endorhizosphere El3E18 23.99 45 SPG Rhizosphere SPG1R5 23.62 46 Laird Rhizosphere La3R9 23.46 47 Carbi Endorhizosphere Ca3E10 22.91 48 Laird Rhizosphere La2R15 22.83 49 Elrose Endorhizosphere El3E14 21.74 50 SPG Rhizosphere SPG2R3 21.63 51 Limerick Endorhizosphere Li3E20 21.17 52 Scott Rhizosphere Sc2R4 21.17 53 Skarsguard Endorhizosphere Sk2E12 21.00 54 Carbi Endorhizosphere Ca2E1 20.47 55 SPG Rhizosphere SPG3R10 20.11 56 Elrose Rhizosphere El1R7 20.04 57 Elrose Endorhizosphere El1E7 19.81 58 Elrose Endorhizosphere El1E4 19.29 59 Elrose Endorhizosphere El3E15 19.25 60 Sutherland Rhizosphere Su2R22 18.99 61 Elrose Rhizosphere El2R9 18.75 62 SPG Rhizosphere SPG1R6 18.65 63 Elrose Endorhizosphere El3E11 18.29 64 Laird Rhizosphere La3R7 18.27 65 Elrose Rhizosphere EL3R9 18.03 66 Sutherland Rhizosphere Su2R20 17.75 67 Sutherland Rhizosphere Su2R12 17.66 68 Sutherland Rhizosphere Su2R17 17.54 69 Elrose Endorhizosphere El3E4 17.27 70 SPG Endorhizosphere SPG3E3 17.25 71 Laird Rhizosphere La2R13 17.19 72 Elrose Endorhizosphere El1E8 17.06 73 Elrose Endorhizosphere El3E16 17.06 74 Limerick Rhizosphere Li3R13 17.05 75 Limerick Rhizosphere Li2R10 16.67 76

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Sutherland Rhizosphere Su2R7 16.42 77 Sutherland Rhizosphere Su2R9 16.36 78 Elrose Endorhizosphere El3E9 16.33 79 Limerick Rhizosphere Li1R10 15.92 80 Elrose Rhizosphere El1R3 15.86 81 Elrose Rhizosphere El2R7 15.80 82 Elrose Endorhizosphere El3E2 15.51 83 Laird Rhizosphere La3R8 15.11 84 Carbi Endorhizosphere Ca2E3 14.93 85 Laird Endorhizosphere La3E14 14.66 86 Elrose Endorhizosphere El3E20 14.53 87 Sutherland Rhizosphere Su2R3 14.43 88 Scott Rhizosphere Sc1R5 14.19 89 Elrose Endorhizosphere El1E3 13.90 90 Scott Rhizosphere Sc3R6 13.87 91 Elrose Endorhizosphere El3E12 13.67 92 Elrose Rhizosphere El2R3 13.47 93 Elrose Rhizosphere El1R1 13.38 94 Carbi Endorhizosphere Ca3E12 13.37 95 Laird Rhizosphere La1R4 13.32 96 Laird Endorhizosphere La3E12 13.24 97 Carbi Endorhizosphere Ca3E6 13.20 98 Elrose Rhizosphere El1R6 12.99 99 Carbi Rhizosphere Ca2R7 12.94 100 SPG Rhizosphere SPG2R7 12.78 101 Carbi Endorhizosphere Ca3E5 12.77 102 Carbi Endorhizosphere Ca3E15 12.76 103 Elrose Endorhizosphere El3E5 12.53 104 Limerick Rhizosphere Li2R8 12.31 105 Elrose Endorhizosphere El3E3 12.28 106 Limerick Rhizosphere Li1R15 12.19 107 Limerick Endorhizosphere Li1E18 12.10 108 Elrose Rhizosphere El1R9 12.09 109 Laird Rhizosphere La1R1 12.02 110 Laird Rhizosphere La2R14 11.95 111 Carbi Endorhizosphere Ca2E11 11.93 112 Scott Rhizosphere Sc3R8 11.88 113 Elrose Endorhizosphere El1E5 11.87 114 Limerick Rhizosphere Li2R6 11.80 115 Carbi Endorhizosphere Ca3E11 11.80 116

104

Sutherland Endorhizosphere Su3E5 11.68 117 Laird Rhizosphere La2R8 11.68 118 Carbi Endorhizosphere Ca3E13 11.66 119 Limerick Endorhizosphere Li1E4 11.64 120 Carbi Endorhizosphere Ca3E7 11.58 121 Limerick Rhizosphere Li2R5 11.53 122 Laird Endorhizosphere La2E8 11.53 123 Laird Rhizosphere La3R11 11.52 124 Elrose Rhizosphere El2R6 11.37 125 Scott Endorhizosphere Sc1E10 11.24 126 Sutherland Rhizosphere Su2R2 11.18 127 Elrose Rhizosphere El2R2 11.17 128 Laird Rhizosphere La1R2 11.15 129 Sutherland Rhizosphere Su2R6 11.06 130 Scott Rhizosphere Sc2R6 11.01 131 Sutherland Endorhizosphere Su3E6 10.97 132 Limerick Endorhizosphere Li1E13 10.92 133 Elrose Rhizosphere El2R4 10.89 134 Scott Rhizosphere Sc3R9 10.86 135 Scott Endorhizosphere Sc3E3 10.86 136 Sutherland Rhizosphere Su2R1 10.79 137 SPG Rhizosphere SPG2R8 10.74 138 Elrose Rhizosphere El3R8 10.72 139 Limerick Endorhizosphere Li1E12 10.60 140 Skarsguard Rhizosphere Sk1R10 10.52 141 Sutherland Rhizosphere Su2R16 10.48 142 Elrose Endorhizosphere El3E1 10.47 143 Laird Rhizosphere La2R12 10.41 144 Sutherland Rhizosphere Su2R11 10.40 145 Scott Rhizosphere Sc3R4 10.40 146 Skarsguard Rhizosphere Sk2R6 10.35 147 Laird Rhizosphere La3R13 10.28 148 Limerick Endorhizosphere Li3E9 10.24 149 Laird Rhizosphere La1R3 10.22 150 Sutherland Endorhizosphere Su3E9 10.16 151 Laird Rhizosphere La3R1 10.15 152 Limerick Endorhizosphere Li3E5 10.07 153 Elrose Endorhizosphere El3E21 10.07 154 Sutherland Rhizosphere Su2R23 10.05 155 Scott Rhizosphere Sc1R8 10.00 156 Scott Rhizosphere Sc3R5 9.97 157 Scott Endorhizosphere Sc1E21 9.97 158 105

Limerick Rhizosphere Li3R12 9.90 159 Laird Rhizosphere La1R6 9.88 160 Scott Endorhizosphere Sc2E10 9.85 161 Sutherland Endorhizosphere Su3E8 9.80 162 Scott Rhizosphere Sc2R5 9.75 163 Sutherland Rhizosphere Su3R8 9.63 164 Limerick Endorhizosphere Li3E8 9.57 165 Scott Rhizosphere Sc2R9 9.52 166 Limerick Endorhizosphere Li1E16 9.51 167 Sutherland Rhizosphere Su3R12 9.51 168 Elrose Rhizosphere El3R1 9.51 169 Scott Endorhizosphere Sc1E9 9.46 170 Skarsguard Rhizosphere Sk1R2 9.43 171 Laird Rhizosphere La2R7 9.40 172 SPG Rhizosphere SPG1R8 9.40 173 Scott Rhizosphere Sc3R3 9.40 174 Scott Endorhizosphere Sc1E11 9.37 175 Skarsguard Rhizosphere Sk1R3 9.33 176 Scott Endorhizosphere Sc2E12 9.27 177 Skarsguard Rhizosphere Sk1R12 9.27 178 Limerick Endorhizosphere Li3E10 9.23 179 SPG Endorhizosphere SPG1E3 9.22 180 Limerick Rhizosphere Li3R5 9.20 181 Scott Endorhizosphere Sc1E20 9.18 182 Limerick Endorhizosphere Li1E11 9.13 183 Scott Rhizosphere Sc3R7 8.99 184 Carbi Rhizosphere Ca3R7 8.97 185 SPG Endorhizosphere SPG2E1 8.97 186 Limerick Rhizosphere Li2R7 8.89 187 Sutherland Rhizosphere Su3R6 8.85 188 Sutherland Endorhizosphere Su3E11 8.66 189 Carbi Endorhizosphere Ca3E4 8.65 190 Sutherland Rhizosphere Su2R15 8.58 191 Laird Endorhizosphere La3E11 8.51 192 Limerick Rhizosphere Li3R6 8.50 193 Scott Endorhizosphere Sc1E3 8.45 194 Sutherland Endorhizosphere Su3E7 8.42 195 Skarsguard Rhizosphere Sk1R1 8.39 196 Laird Rhizosphere La1R7 8.27 197 Scott Endorhizosphere Sc1E18 8.24 198 Limerick Rhizosphere Li2R4 8.07 199

106

Sutherland Rhizosphere Su3R9 8.04 200 Limerick Rhizosphere Li1R12 7.96 201 Limerick Endorhizosphere Li1E1 7.95 202 Laird Rhizosphere La3R5 7.95 203 Carbi Rhizosphere Ca3R5 7.92 204 Scott Endorhizosphere Sc1E5 7.91 205 Carbi Rhizosphere Ca1R2 7.80 206 Sutherland Endorhizosphere Su3E10 7.76 207 Elrose Rhizosphere El3R10 7.75 208 Limerick Rhizosphere Li2R9 7.73 209 Scott Endorhizosphere Sc1E19 7.66 210 Scott Endorhizosphere Sc1E7 7.63 211 Scott Endorhizosphere Sc1E15 7.63 212 Scott Endorhizosphere Sc1E1 7.60 213 Elrose Rhizosphere El2R1 7.59 214 Scott Endorhizosphere Sc1E4 7.54 215 Elrose Rhizosphere El3R2 7.51 216 Scott Endorhizosphere Sc1E14 7.49 217 Skarsguard Endorhizosphere Sk3E10 7.44 218 Elrose Rhizosphere El2R8 7.43 219 Laird Endorhizosphere La1E8 7.43 220 Carbi Rhizosphere Ca2R5 7.41 221 Limerick Rhizosphere Li2R2 7.38 222 Laird Rhizosphere La2R2 7.32 223 Laird Rhizosphere La3R15 7.31 224 Limerick Endorhizosphere Li3E14 7.31 225 Elrose Rhizosphere El1R12 7.29 226 Scott Rhizosphere Sc3R2 7.26 227 Sutherland Rhizosphere Su3R2 7.26 228 Elrose Rhizosphere El1R10 7.23 229 Laird Endorhizosphere La2E4 7.22 230 Carbi Endorhizosphere Ca3E2 7.20 231 Elrose Endorhizosphere El1E1 7.20 232 Limerick Endorhizosphere Li3E25 7.19 233 Carbi Rhizosphere Ca1R7 7.10 234 Scott Endorhizosphere Sc1E16 7.10 235 Limerick Rhizosphere Li3R11 7.10 236 Scott Endorhizosphere Sc1E2 7.02 237 Laird Endorhizosphere La1E3 7.01 238 Laird Endorhizosphere La1E13 6.97 239 Limerick Endorhizosphere Li3E26 6.95 240

107

Laird Endorhizosphere La1E14 6.93 241 Skarsguard Endorhizosphere Sk2E11 6.92 242 Limerick Endorhizosphere Li3E7 6.91 243 Limerick Endorhizosphere Li3E23 6.85 244 Elrose Rhizosphere El1R2 6.84 245 Scott Rhizosphere Sc2R7 6.83 246 Carbi Rhizosphere Ca1R5 6.83 247 Laird Rhizosphere La1R8 6.82 248 Carbi Rhizosphere Ca1R9 6.79 249 Elrose Endorhizosphere El1E9 6.73 250 Scott Endorhizosphere Sc1E17 6.72 251 Scott Endorhizosphere Sc1E13 6.66 252 Laird Rhizosphere La1R5 6.65 253 Skarsguard Endorhizosphere Sk3E3 6.62 254 Limerick Endorhizosphere Li3E6 6.57 255 Scott Endorhizosphere Sc1E8 6.56 256 Carbi Endorhizosphere Ca2E4 6.51 257 Laird Rhizosphere La3R12 6.49 258 Elrose Rhizosphere El3R11 6.43 259 Limerick Endorhizosphere Li3E17 6.42 260 Skarsguard Endorhizosphere Sk2E10 6.39 261 Laird Rhizosphere La3R10 6.37 262 Elrose Rhizosphere El1R4 6.29 263 Limerick Endorhizosphere Li3E12 6.25 264 Laird Rhizosphere La1R9 6.22 265 Scott Rhizosphere Sc1R2 6.22 266 Laird Rhizosphere La2R3 6.21 267 Laird Endorhizosphere La1E17 6.21 268 Limerick Endorhizosphere Li3E1 6.19 269 Limerick Endorhizosphere Li3E3 6.13 270 Limerick Rhizosphere Li3R2 6.13 271 Scott Endorhizosphere Sc2E1 6.13 272 Skarsguard Endorhizosphere Sk2E3 6.11 273 Laird Rhizosphere La3R16 6.10 274 Scott Rhizosphere Sc1R1 6.10 275 Limerick Rhizosphere Li2R1 6.10 276 Limerick Rhizosphere Li3R1 6.10 277 Limerick Endorhizosphere Li3E15 6.09 278 Limerick Rhizosphere Li2R3 6.07 279 Limerick Endorhizosphere Li2E5 6.04 280 Scott Rhizosphere Sc3R1 5.97 281

108

Scott Endorhizosphere Sc1E12 5.94 282 Limerick Rhizosphere Li1R6 5.91 283 Laird Rhizosphere La2R9 5.90 284 Carbi Rhizosphere Ca2R9 5.85 285 Scott Rhizosphere Sc1R3 5.80 286 Sutherland Rhizosphere Su3R7 5.79 287 Elrose Rhizosphere El2R5 5.79 288 Carbi Rhizosphere Ca1R4 5.76 289 Laird Endorhizosphere La3E5 5.73 290 Limerick Endorhizosphere Li3E2 5.69 291 Skarsguard Rhizosphere Sk3R8 5.66 292 Carbi Endorhizosphere Ca3E3 5.64 293 Limerick Endorhizosphere Li3E22 5.62 294 Limerick Rhizosphere Li3R14 5.62 295 Scott Endorhizosphere Sc2E3 5.61 296 Laird Rhizosphere La3R6 5.61 297 Limerick Rhizosphere Li2R11 5.61 298 Laird Rhizosphere La2R11 5.59 299 Skarsguard Endorhizosphere Sk3E15 5.58 300 Carbi Rhizosphere Ca3R2 5.55 301 Laird Endorhizosphere La3E3 5.53 302 Limerick Rhizosphere Li3R7 5.51 303 Skarsguard Rhizosphere Sk1R6 5.50 304 Skarsguard Endorhizosphere Sk2E2 5.49 305 Laird Endorhizosphere La1E1 5.49 306 Carbi Rhizosphere Ca1R6 5.48 307 Skarsguard Rhizosphere Sk3R9 5.48 308 Elrose Endorhizosphere El1E2 5.46 309 Limerick Endorhizosphere Li3E16 5.45 310 Limerick Rhizosphere Li3R9 5.42 311 Carbi Rhizosphere Ca3R9 5.36 312 Elrose Rhizosphere El3R7 5.35 313 Limerick Endorhizosphere Li3E13 5.34 314 Carbi Endorhizosphere Ca2E10 5.33 315 Laird Endorhizosphere La3E4 5.31 316 Limerick Endorhizosphere Li3E27 5.30 317 Skarsguard Rhizosphere Sk3R7 5.30 318 Laird Rhizosphere La2R1 5.29 319 Limerick Endorhizosphere Li2E6 5.29 320

109

Skarsguard Rhizosphere Sk3R6 5.28 321 Limerick Endorhizosphere Li3E24 5.27 322 Skarsguard Rhizosphere Sk1R5 5.23 323 Limerick Endorhizosphere Li2E8 5.23 324 Limerick Endorhizosphere Li2E4 5.19 325 Laird Endorhizosphere La1E2 5.10 326 Carbi Endorhizosphere Ca2E7 5.09 327 Skarsguard Rhizosphere Sk2R9 5.09 328 Scott Endorhizosphere Sc1E6 5.03 329 Carbi Endorhizosphere Ca2E9 5.03 330 Limerick Endorhizosphere Li3E4 5.00 331 Carbi Endorhizosphere Ca3E1 4.97 332 Skarsguard Endorhizosphere Sk3E4 4.96 333 Elrose Endorhizosphere El2E11 4.90 334 Elrose Endorhizosphere El2E1 4.90 335 Laird Endorhizosphere La1E7 4.89 336 Limerick Endorhizosphere Li1E7 4.82 337 Scott Rhizosphere Sc1R4 4.76 338 Laird Endorhizosphere La3E1 4.76 339 Skarsguard Rhizosphere Sk1R11 4.75 340 Laird Endorhizosphere La3E2 4.74 341 Carbi Endorhizosphere Ca2E2 4.73 342 Limerick Endorhizosphere Li2E3 4.72 343 Elrose Endorhizosphere El2E13 4.69 344 Carbi Rhizosphere Ca1R1 4.69 345 Scott Endorhizosphere Sc2E4 4.68 346 Carbi Endorhizosphere Ca2E12 4.67 347 Limerick Rhizosphere Li3R10 4.66 348 Skarsguard Endorhizosphere Sk3E13 4.65 349 Scott Rhizosphere Sc1R9 4.64 350 Carbi Rhizosphere Ca1R12 4.63 351 Elrose Endorhizosphere El2E4 4.62 352 Skarsguard Rhizosphere Sk2R5 4.56 353 Skarsguard Rhizosphere Sk2R8 4.54 354 Limerick Endorhizosphere Li3E11 4.48 355 Carbi Endorhizosphere Ca1E1 4.47 356 Scott Endorhizosphere Sc2E7 4.46 357 Limerick Endorhizosphere Li2E2 4.45 358 Laird Endorhizosphere La3E6 4.40 359 Carbi Rhizosphere Ca1R3 4.35 360 Sutherland Rhizosphere Su3R11 4.35 361 Laird Rhizosphere La3R14 4.31 362 110

Skarsguard Rhizosphere Sk1R4 4.30 363 Limerick Endorhizosphere Li1E6 4.29 364 Limerick Endorhizosphere Li1E8 4.26 365 Limerick Rhizosphere Li3R3 4.26 366 Sutherland Rhizosphere Su3R5 4.23 367 Scott Endorhizosphere Sc2E8 4.23 368 Carbi Endorhizosphere Ca2E5 4.19 369 Elrose Endorhizosphere El3E7 4.15 370 Scott Endorhizosphere Sc2E2 4.14 371 Skarsguard Endorhizosphere Sk3E5 4.14 372 Limerick Rhizosphere Li3R4 4.14 373 Scott Rhizosphere Sc1R6 4.12 374 Skarsguard Endorhizosphere Sk3E9 4.12 375 Elrose Endorhizosphere El3E8 4.10 376 Limerick Endorhizosphere Li1E15 4.05 377 Skarsguard Endorhizosphere Sk3E12 4.05 378 Skarsguard Rhizosphere Sk2R2 4.02 379 Laird Endorhizosphere La3E9 3.98 380 Elrose Endorhizosphere El2E8 3.92 381 Limerick Endorhizosphere Li3E18 3.91 382 Skarsguard Endorhizosphere Sk3E14 3.89 383 Scott Rhizosphere Sc1R7 3.88 384 Carbi Rhizosphere Ca3R1 3.86 385 Limerick Rhizosphere Li2R12 3.86 386 Laird Endorhizosphere La1E15 3.83 387 Limerick Rhizosphere Li1R1 3.83 388 Laird Endorhizosphere La2E2 3.78 389 Laird Rhizosphere La2R10 3.74 390 Carbi Endorhizosphere Ca1E3 3.72 391 Carbi Endorhizosphere Ca3E8 3.72 392 Carbi Rhizosphere Ca3R4 3.68 393 Elrose Endorhizosphere El2E15 3.64 394 Carbi Rhizosphere Ca2R4 3.58 395 Elrose Endorhizosphere El2E5 3.46 396 Skarsguard Endorhizosphere Sk3E18 3.44 397 Elrose Endorhizosphere El2E14 3.44 398 Limerick Endorhizosphere Li1E9 3.43 399 Carbi Endorhizosphere Ca3E20 3.40 400 Limerick Rhizosphere Li1R2 3.34 401 Elrose Endorhizosphere El2E3 3.34 402 Elrose Endorhizosphere El2E9 3.32 403

111

Scott Endorhizosphere Sc2E9 3.31 404 Limerick Endorhizosphere Li2E1 3.29 405 Carbi Endorhizosphere Ca1E2 3.27 406 Elrose Endorhizosphere El3E6 3.21 407 Carbi Endorhizosphere Ca2E8 3.19 408 Limerick Rhizosphere Li1R3 3.16 409 Skarsguard Endorhizosphere Sk3E2 3.13 410 SPG Rhizosphere SPG1R7 3.08 411 Skarsguard Endorhizosphere Sk3E17 2.99 412 Skarsguard Endorhizosphere Sk2E1 2.97 413 Laird Endorhizosphere La3E16 2.92 414 Limerick Rhizosphere Li1R13 2.90 415 Laird Endorhizosphere La2E1 2.88 416 Laird Endorhizosphere La1E21 2.83 417 Elrose Endorhizosphere El2E6 2.79 418 Laird Endorhizosphere La1E19 2.75 419 Carbi Rhizosphere Ca2R6 2.67 420 Skarsguard Endorhizosphere Sk3E16 2.65 421 Elrose Endorhizosphere El2E7 2.60 422 Carbi Rhizosphere Ca2R8 2.57 423 Carbi Rhizosphere Ca3R8 2.52 424 Sutherland Rhizosphere Su2R13 2.39 425 Sutherland Rhizosphere Su3R1 2.39 426 Laird Rhizosphere La3R18 2.32 427 Laird Endorhizosphere La1E20 2.30 428 Elrose Endorhizosphere El2E2 2.27 429 Sutherland Rhizosphere Su3R3 2.17 430 Laird Endorhizosphere La3E15 2.10 431 Elrose Rhizosphere El1R11 2.02 432 Carbi Rhizosphere Ca2R2 1.74 433 Carbi Rhizosphere Ca1R8 1.59 434 Sutherland Rhizosphere Su1R3 1.55 435 Sutherland Rhizosphere Su1R11 1.55 436 Elrose Endorhizosphere EL1E6 1.54 437 Elrose Rhizosphere EL3R12 1.45 438 SPG Rhizosphere SPG3R13 1.31 439 SPG Rhizosphere SPG3R11 1.30 440 SPG Endorhizosphere SPG2E3 1.27 441

112

SPG Endorhizosphere SPG3E5 1.25 442 Sutherland Rhizosphere Su1R15 1.23 443 SPG Rhizosphere SPG3R1 1.22 444 SPG Rhizosphere SPG3R2 1.13 445 SPG Rhizosphere SPG3R3 1.13 446 SPG Endorhizosphere SPG2E5 1.13 447 Sutherland Rhizosphere Su1R4 1.04 448 Elrose Rhizosphere EL3R6 0.92 449 SPG Endorhizosphere SPG2E4 0.85 450 SPG Rhizosphere SPG3R7 0.80 451 SPG Rhizosphere SPG3R8 0.80 452 SPG Rhizosphere SPG3R4 0.79 453 SPG Rhizosphere SPG3R5 0.79 454 SPG Endorhizosphere SPG3E2 0.77 455 SPG Endorhizosphere SPG1E1 0.76 456 SPG Endorhizosphere SPG2E6 0.63 457 SPG Endorhizosphere SPG3E6 0.56 458 SPG Rhizosphere SPG3R6 0.17 459 SPG Endorhizosphere SPG3E1 0.06 460 SPG Endorhizosphere SPG2E2 0.05 461 SPG Rhizosphere SPG3R12 0.04 462 Carbi Rhizosphere Ca2R3 BDLND 463 Carbi Rhizosphere Ca3R6 BDLND 464 Elrose Rhizosphere El3R5 BDLND 465 Limerick Rhizosphere Li3R15 BDLND 466 Limerick Rhizosphere Li3R8 BDLND 467 SPG Endorhizosphere SPG3E4 BDLND 468 Sutherland Rhizosphere Su1R10 BDLND 469 Sutherland Rhizosphere Su1R12 BDLND 470 Sutherland Rhizosphere Su1R17 BDLND 471 Sutherland Rhizosphere Su1R19 BDLND 472 Sutherland Rhizosphere Su1R21 BDLND 473 Sutherland Rhizosphere Su1R5 BDLND 474 Sutherland Rhizosphere Su1R6 BDLND 475 Sutherland Rhizosphere Su1R7 BDLND 476 Sutherland Rhizosphere Su1R9 BDLND 477 † ND= Not detected.

113

Table A.3 Shoot biomass, nutrient concentration and total uptake of lentil (cv. CDC Milestone) grown for 45 days in a growth chamber and inoculated with siderophore-producing bacteria. Results are average of 4 replications. ------Concentration------Total uptake------Isolates Shoot (g) N (g·kg-1) P (g·kg-1) Fe (mg·kg-1) N (mg·pot-1) P (mg·pot-1) Fe(µg·pot-1) Ca2R1 1.30 a 27.25 abc 1.44 ab 42.50 d 32.22 abc 1.86 abc 55 de Control 0.83 f 21.90 c 1.16 b 57.60 cd 18.86 abc 1.00 cdef 48 e El1R5 0.64 hijkl 33.28 ab 1.15 b 103.90 bcd 30.87 abc 1.07 bcdef 94 b El1R8 0.93 e 26.34 abc 1.18 b 158.50 bcd 26.28 abc 1.16 abcdef 155 a El3E13 0.89 ef 25.77 abc 1.16 b 64.30 cd 24.12 abc 1.04 cdef 57 de La3E4 0.77 fghij 27.54 abc 1.11 b 60.25 cd 25.51 abc 1.08 cdef 46 b La3E13 0.58 ijkl 29.47 abc 1.18 b 147.90 bcd 18.90 abc 0.73 ef 91 e La3R2 0.81 f 32.81 ab 0.98 b 163.10 bcd 26.25 abc 0.77 def 128 a La3R3 0.88 ef 27.98 abc 1.18 b 159.60 bcd 20.49 abc 0.74 ef 99 ab Li1R7 1.22 ab 27.69 abc 1.40 ab 87.08 cd 26.67 abc 1.34 abcdef 106 ab Li1R8 1.16 bc 25.06 abc 1.65 ab 56.45 cd 34.82 abc 1.83 abcd 65 cd Li3E19 0.50 kl 31.72 abc 1.45 ab 276.50 a 15.63 bc 0.68 ef 137 a Li3E21 1.00 bcdef 24.20 abc 0.98 b 53.70 d 24.75 abc 1.00 cdef 53 e Sc2R1 1.02 bcde 28.25 abc 1.01 b 134.70 bcd 28.80 abc 1.04 bcdef 136 a Sc2R10 0.74 fghijkl 23.67 bc 1.04 b 96.60 cd 20.33 abc 0.88 cdef 80 bc Sc2R11 0.93 e 24.02 bc 1.15 b 95.99 cd 25.15 abc 1.02 cdef 89 b Sc2R12 1.21 ab 25.10 abc 1.22 b 96.00 cd 31.67 abc 1.55 abcdef 119 a Sc2R2 1.34 a 21.01 c 1.08 b 64.10 cd 28.66 abc 1.44 abcdef 84 bc Sc2R3 0.43 l 28.26 abc 1.67 ab 86.80 cd 12.37 c 0.72 ef 37 f Sk2R3 0.61 hijkl 27.38 abc 1.55 ab 125.36 bcd 10.37 c 0.69 ef 76 bc Sk2R4 0.44 l 28.84 abc 1.53 ab 120.00 bcd 11.54 c 0.61 f 47 e Sk3R1 0.89 ef 29.65 abc 1.58 ab 72.68 cd 29.08 abc 1.68 abcde 65 cd Sk3R2 0.73 fghijkl 28.23 abc 1.26 b 183.20 bc 18.28 abc 0.81 cdef 115 ab Sk3R3 1.22 ab 32.78 abc 1.74 ab 86.53 cd 42.83 a 2.20 a 105 ab Sk3R4 1.08 abcde 37.49 a 1.68 ab 93.60 cd 38.43 ab 1.60 abcdef 101 ab SPG1R1 0.90 ef 25.09 abc 1.12 b 118.30 bcd 22.30 abc 0.99 cdef 102 ab SPG1R2 0.92 ef 28.69 abc 1.21 b 92.20 cd 26.02 abc 1.10 bcdef 83 bc SPG1R3 0.77 fghij 29.13 abc 1.43 ab 141.50 bcd 23.88 abc 1.14 abcdef 115 ab SPG1R4 0.76 fghijk 28.89 abc 1.25 b 108.10 bcd 19.25 abc 0.80 def 71 bcd SPG2R10 0.84 f 32.96 ab 1.32 b 140.40 bcd 21.26 abc 0.84 cdef 87 bc SPG2R11 0.49 kl 35.39 a 1.46 ab 224.40 ab 17.30 bc 0.70 ef 111 ab SPG2R12 0.46 kl 31.61 abc 1.60 ab 70.95 cd 14.84 bc 0.74 def 33 f SPG2R4 0.62 hijkl 33.43 ab 1.49 ab 102.50 bcd 16.87 bc 0.74 ef 50 e SPG2R5 0.70 fghijkl 26.92 abc 1.24 b 103.50 bcd 20.67 abc 0.92 cdef 78 bc SPG2R6 1.13 c 29.72 abc 1.27 b 74.10 cd 25.91 abc 1.13 bcdef 69 bcd SPG3R9 0.66 ghijkl 30.21 abc 1.23 b 148.30 bcd 21.18 abc 0.86 cdef 102 b Su2R10 0.74 fghijk 29.63 abc 1.04 b 83.70 cd 22.43 abc 0.79 def 62 ab Su2R18 0.55 jkl 32.35 abc 1.56 ab 135.70 bcd 18.36 abc 0.88 cdef 76 cd Su2R21 1.33 a 28.01 abc 1.57 ab 63.68 cd 39.36 ab 2.10 ab 84 bc Su2R24 0.72 fghijkl 31.26 abc 1.49 ab 77.37 cd 24.23 abc 1.15 bcdef 56 de Su2R5 0.42 l 35.54 a 2.14 a 71.80 cd 13.03 c 0.78 def 30 f Different letters in the same column represent significant differences at the level of 5%.

114

Table A.4 Correlation coefficients among plant nutrient parameters and bacterial siderophore concentration in lentil plants at 45 DAP

------Concentration------Total uptake------†Siderophore Shoot N P Fe N P Fe concentration Shoot 1

N -0.43 *** 1

Concentration P -0.21 ** 0.52 *** 1

Fe -0.47 *** 0.36 *** -0.13 * 1

N 0.89 *** -0.06 *** -0.01 *** -0.36 1

Total uptake P 0.86 *** -0.16 0.26 *** -0.49 *** 0.90 *** 1

Fe 0.18 * 0.09 *** -0.37 0.73 *** 0.24 * 0.02 * 1

†Siderophore -0.28 -0.10 0.08 -0.01 -0.34 -0.20 -0.26 1 concentration

†Siderophore concentration measured in vitro.

115 Significant levels: ‘***’ 0.001; ‘**’ 0.01; ‘*’ 0.05

115

Table A.5 ANOVA analysis of biomass, nutrient concentration and uptake by pea plants at 45 DAP inoculated with siderophore- producing bacteria.

------Concentration------Total uptake------Shoot N (g·kg-1) P (g·kg-1) Fe (mg·kg-1) N (mg·pot-1) P (mg·pot-1) Fe(µg·pot-1)

Variety <0.0001 0.0212 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

Isolate <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

Variety × Isolate <0.0001 <0.0001 <0.0001 0.0031 <0.0001 <0.0001 <0.0001 P values < 0.01 are significant at 1 % level. 116

116

Table A.6 Pea biomass, total-N, -P and -Fe contents of plants inoculated with siderophore-producing bacteria, grown in a Brown potted soil (Ardil association) for 45 DAP in a growth chamber. Results are average of 3 replications. 117

Different letters in the same column indicate significant differences at α=0.05, using Tukey’s post hoc test.

117