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STUDYING THE GENTIC BASIS OF PLANT-MICROBE INTERACTION FOR CROP
IMPROVEMENT Commented [AS1]: Cover Page

……………………………………………………………………………………………………..

AFOLABI, SAMUEL BAMIDELE

MCB/2019/1071

DEPARTMENT OF MICROBIOLOGY

FACULTY OF SCIENCE

FEDERAL UNIVERSITY OYE-EKITI,

EKITI STATE

AUGUST, 2024
 STUDYING THE GENETIC BASIS OF PLANT-MICROBE INTERACTIONS
FOR CROP IMPROVEMENT Commented [AS2]: Title page

BY

AFOLABI, SAMUEL BAMIDELE

MCB/2019/1071

SUBMITTED TO

DEPARTMENT OF MICROBIOLOGY

FACULTY OF SCIENCE

FEDERAL UNIVERSITY OYE-EKITI,

EKITI STATE, NIGERIA.

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF

THE DEGREE OF BACHELOR OF SCIENCE (B.Sc) IN MICROBIOLOGY

AUGUST, 2024
 CERTIFICATION

This is to certify that AFOLABI, SAMUEL BAMIDELE, bearing matriculation number

MCB/2019/1071, under the supervision of Prof. Mrs. O. M. Oyawoye in the Department of

Microbiology, Federal University Oye-Ekiti, Nigeria.

AFOLABI, SAMUEL BAMIDELE __________________

MCB/2019/1071 SIGNATURE / DATE

PROF. O. M., OYAWOYE __________________

SUPERVISOR SIGNATURE / DATE

DR. S. K., OJO __________________

Ag. HOD, MICROBIOLOGY DEPARTMENT SIGNATURE / DATE

i
 DEDICATION

This report is dedicated to God Almighty, my late father Mr. Wale Afolabi for his prayer and love

over me.

ii
 ACKNOWLEDGEMENTS

I acknowledged my unique supervisor Prof. Mrs. O. M., Oyawoye for her advice and lesson, my

Co-supervisor, Dr. Oluwatosin A., Ajibade for his lesson and coaching.

I acknowledged my wonderful Ag. H.O.D, Dr. S. K., OJO and all the lecturers in Microbiology

department.

I appreciate my beloved mother Mrs. Mary Afolabi and siblings for their prayer and

encouragement. I also appreciate my loving uncle, Mr. Wale Daramola for his financial support

and prayers, and to my darling friend, Ajibola Adesola P., Ikotun Abosede M. and to my fellow

colleagues for their massive support and contribution for corrections which sum up to make this a

wonderful experience, Thank you.

iii
 TABLE OF CONTENT

Title page

Certification i

Dedication ii

Acknowledgement iii

Table of Content iv-ix

List of Plates x

List of Tables xi

List of Maps and Figures xii

Abstract xiii

Chapter One

1.0. Introduction

1.1.Background Study 1

1.2.Statement of Problem 4

1.3.Justification of Study 4

1.4.Aim And Objectives of the Study 5

1.4.1. Aim of The Study 5

1.4.2. Objectives of the Study 6

1.5.Research Questions 6

Chapter Two

2.0. Literature Review 7

iv
2.1.Beneficial Plant-Microbe Interaction 7

2.2. Direct Mechanism of Crop Improvement by Microbes 8

2.2.1. Nutrient Acquisition and Growth Promotion 8

2.2.2. Production Of Siderophore and Osmotic Balance 10

2.2.3. Phytohormone Production 11

2.3. Indirect Mechanism 13

2.3.1. Stress Mitigation 13

2.3.2. Antibiotic Production 14

2.3.3. Hydrolytic Enzymes Production 14

2.4. Root Exudates and Signalling 15

2.5. Plant-Microbe Molecular Complex 18

2.5.1. Recognition and Signal Transmission 20

2.5.2. Environmental Effects and Microbial Signals in Plant Defense 21

2.5.3. Chemotaxis and Quorum Sensing 24

2.5.4. Phytohormone Signaling 25

2.5.5. Secondary Metabolite Production 28

2.6. Genomic And Proteomics Approach for Plant-Microbe Interaction. 28

2.7. Transcription Factors in Plant–Microbe Interactions 32

v
Chapter Three

3.0. Materials And Methods 35

3.0. Area Of Study 35

3.1. Soil Sample Collection 35

3.1.1. Soil Sampling for Metagenomics Analysis 37

3.1.2. Soil Sample Analysis 37

3.2. Isolation And cfu Determination of Bacterial Strains 37

3.2.1. Purification (Subculturing) of Bacterial Isolates 38

3.3. Gram's Staining Technique 39

3.5 Biochemical Characterization of the Isolates 35

3.5.1 Catalase Test 39

3.5.2 Coagulase Test 40

3.5.3 Indole Test 40

3.5.4 Oxidase Test 41

3.5.5 Sugar Fermentation Test 41

3.5.6 Starch Hydrolysis Test 42

3.5.7 Citrate Test 42

vi
3.5.8 Sulphur Reducing Test 43

3.5.9 MR-VP Test (Methyl Red Vouges Pros Kaver Test) 43

3.6 Screening Bacterial Isolates for Plant Growth-Promoting Activity 44

3.6.1 Ammonia Production Test 44

3.6.2 Auxin (IAA) Production Assay 44

3.6.3 Phosphate Solubilization 45

3.6.4 Protease And α- Amylase Production 46

3.7 Molecular Identification of The Selected PSB And Metagenomics 47

3.7.1 Bacterial DNA Extraction 47

3.7.2 Polymerase Chain Reaction 48

3.7.3 Electrophoresis 48

3.7.4 Illumina Sequencing Method 49

3.7.5 Comprehensive and Functional Genome Analysis of

Acinetobacter baumannii strain OYA S31 50

3.8 Field Trial within Controlled Greenhouse Environment 50

3.8.1 Maize Seed Bacterization 50

3.8.2 Plant Experiment 51

3.9 Statistical Analysis of Measurement 52

vii
Chapter Four

4.0. Result

4.1. Colony Count and Morphological Characteristics of Bacterial Isolates 53

4.2. Gram Staining and Biochemical Characteristics of Bacterial Isolates 53

4.3. Soil Agronomic Parameters (Results for Soil Analysis) 54

4.4. Screening for Plant Growth Promoting Activities 60

4.4.1. Growth Promoting Trait: Auxin Production Assay of PGPR 60

4.4.2. Nutrient Solubilization Trait: Phosphate Solubilization Assay of PGPR 60

4.4.3. Pectinase Production 60

4.5. DNA Extraction Yield 68

4.6. Comprehensive And Functional Genome

of Acinetobacter baumannii OYA S31 68

4.6.1. Genome Assembly Features 68

4.6.2. Genome Annotation Features for OYA S31 71

4.6.3. Subsystem Analysis 74

4.6.4. Phylogenetic Analysis 74

4.7. Effects of IS-13 Isolate and OYA S31 on Maize Growth (Zea mays) 77

viii
4.7.1. Plant Height and Girth 81

4.7.2. Root Length and Branching 81

4.7.3. Leaf Morphology 84

Chapter Five

5.0 Discussion, Conclusion and Recommendations

5.1. Discussion 87

5.2. Conclusion 89

5.3. Recommendations 90

References 91

Appendices 116

ix
 LIST OF PLATES

Plate 2.1: Plant-microbiome interactions

Plate 2.2: Microbial pathogens impact host plants, prompting them to emit volatile compounds.

Plate 4.1: Microscopic examination of bacterial isolates after gram staining, viewed under a

magnification of X100.

Plate 4.2.: Phosphate solubilization

Plate 4.3: Production of protease enzyme by bacteria cells on skimmed milk agar.

Plate 4.4: Image of Plant for the Treatments (A=Control, B= OYA S31, C= IS-13 Isolate)

x
 LIST OF TABLES

Table 2.1: Microbial-mediated interactions with host plants

Table 4.1: Total Viable Bacteria Count

Table 4.2: Morphological Characteristics of Bacterial Isolates on Nutrient Agar Plates

Table 4.3: Gram Staining and Biochemical Characteristics of Bacterial Isolates

Table 4.4: Sugar Fermentation Result and Gas Production

Table 4.5: Soil Parameters Result

Table 4.6: Auxin Production Result

Table 4.7: Phosphate Solubilization

Table 4.8: Pectinase Production Result

Table 4.9: DNA Extract yield and purity level

Table 4.10: Assembly Features

Table 4.11: Annotated Genome Features

Table 4.12: Protein Features

Table 4.13: Agronomical parameter of the Maize plant

Table 4.14: Mean and Standard Deviation statistics for the Treatment.

Table 4.15: Influence of Auxin-Producing and Phosphate Solubilizing Bacteria (IS-13) on Maize

Growth Factors

xi
 LIST OF MAPS AND FIGURES

Map 1: Map showing the area of study

Map 2: Map showing where samples are collected.

Figure 4.1: A stacked bar chart depicting the solubilization of phosphate intensity produced by

each PGPR isolate

Figure 4.2: Subsystem pathways

Figure 4.3. Phylogenetic Tree Analysis

Figure 4.4: Bar Chart depicting the average value of Plant girth and Shoot length for each

treatment

Figure 4.5: Bar Chart depicting the average value of Root length and Leaf morphology for each

treatment

Figure 4.6: A stacked bar chart depicting the effect of the IS-13 isolate on Plant Metrics.

xii
 ABSTRACT

Understanding and utilizing Plant Growth Promoting Rhizobacterial (PGPR) can meet the

increasing food demands of a growing global population by enhancing crop yields through

molecular interactions. This research focuses on exploring plant-microbe interactions to advance

crop enhancement using Maize (Zea mays) as the case study. A total of 25 bacteria were isolated

from the rhizosphere of Ananas comosus (pine-apple) on a bearing of 7°46'36.1"N latitude

5°18'43.5"E longitude and assessed for their ability to solubilize phosphate and produce indole

acetic acid. The best 5 organisms with a strong plant growth promoting activity and testing positive

to indole acetic acid production with solubilization index ranging from 3 ± 0.02 to 8 ± 0.10 PSI

were used in conducting this research. IS-13 suspected to be a Pseudomonas fluorescens, a PGPR

active organism with high phosphate solubilizing index of 8 ± 1.95 and auxin production. The IS-

13 was bio-primed with Zea mays (maize) seed and grown in a controlled green house, comparing

to a known PGPR strain, Acinetobacter baumannii OYA S31, and a control treatment. Different

plant parameters such as shoot length, root length and branching, leaf morphology and plant girth

were collected and their effect were compared for each treatment for a period of seven weeks. The

mean values obtained from shoot length are 15.1143 ± 2.53 for IS-13, 12.6429 ± 1.98 for OYA

S31 and 12.2143 ± 1.55 for CONTROL. The mean values obtained for root length over the seven

weeks of measurement are 16.5 ± 1.94 for IS-13, 14.1714 ± 1.48 for OYA S31 and 12.7 ± 0.68 for

Control. The IS-13 significantly increases the maize plant growth by 38% aiding the rapid growth

in the shoot and root sustainability via genetic interaction. The statistical analysis revealed at a

significance threshold of p ≤ 0.05 that the organism IS-13 has strong potential as a PGPR and

biofertilizer, making it a viable option for sustainable agriculture to support the growing population.

xiii
 CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND STUDY

With the global population projected to surpass 9 billion in the next thirty years, a key issue is how

to satisfy the growing demand for food. While chemical fertilizers have significantly boosted crop

yields over the past fifty years, their heavy and prolonged use has led to numerous environmental

issues, including various forms of pollution, ecosystem contamination, and a decline in soil quality

and biodiversity (Majeed et al., 2018). For example, the buildup of synthetic pesticides in the soil

can degrade soil quality, hinder plant growth, or render the plants unfit for consumption (Pascale

et al., 2020). Concurrently, the production of staple crops like maize must rise substantially in the

coming decades to meet the demands of a growing global population. Therefore, a sustainable

agriculture system is critically needed, particularly for primary crops. Although crop improvement

programs are in progress, there has been inadequate focus on modern techniques and balanced

fertilization, resulting in nutritional insecurity in staple crops (Jalal et al., 2024). The World Health

Organization emphasizes the importance of food safety and calls for measures to prevent

foodborne diseases at all stages of food processing, production, storage, transportation, and

consumption (Su et al., 2024). Addressing these challenges is essential for the sustainability of

global food systems, environmental stability, and climate resilience. One emerging approach to

support sustainable agriculture involves using inoculants made with Plant Growth-Promoting

Bacteria (PGPB) (Ullah et al., 2020). PGPB can directly enhance plant growth by aiding the uptake

of soil nutrients through biological nitrogen fixation or the mineralization/solubilization of

phosphate. They also provide phytohormones to plants and protect them from biotic and abiotic

1
stresses. Additionally, they offer indirect benefits by priming plants' resistance responses to stress

(Trivedi et al., 2020). Research in recent decades has uncovered microorganisms associated with

different plants and specific plant parts. Known collectively as the plant holobiont or plant

microbiota, these microorganisms inhabit the rhizosphere, phyllosphere, and endosphere of plants.

The plant microbiota is crucial for promoting plant growth and maintaining plant health (Compant

et al., 2019), playing a role in nutrient acquisition, pathogen protection, and tolerance to abiotic

stress (Dini-Andreote et al., 2014).

Plant growth-promoting rhizobacteria (PGPR) are vital in agriculture, serving as biofertilizers,

biostimulants, and bioprotectants. PGPR generate phytohormones that aid in plant growth and

provide protection against a range of plant pathogens (Wang et al., 2017). They directly promote

plant growth through biological nitrogen fixation, phosphorus uptake, and the production of

phytohormones such as indole-3-acetic acid (IAA), gibberellic acid (GA), and cytokinins (Santner

et al., 2009). Indirectly, PGPR microbes combat plant pathogenic microbes by producing

antibiotics and stimulating induced systemic resistance in plants (Prabhukarthikeyan et al., 2018).

Conversely, numerous plant pathogenic (PP) microorganisms cause severe diseases in crops,

significantly reducing crop yields and posing a major threat to global food security (FAO, 2017).

Maize is often used as a model for studying PGPR due to its global significance as a major crop.

Numerous studies have demonstrated that inoculating maize with PGPR can enhance growth and

yield, even under stress conditions like drought. Additionally, certain PGPRs can protect maize

from pathogen attacks through mechanisms such as antibiosis (producing diffusible and volatile

organic compounds), competition for space, nutrient deprivation, 1-amino cyclopropane-1-

carboxylic acid deaminase activity, and boosting immune defense mechanisms.

2
Another interesting area of analysis in this study is the effect of PGPR inoculation on the assembly

and growth promotion associated with maize recommending that similar studies be expanded to

different environmental settings. Despite numerous studies on the maize microbiome, the effects

of PGPR inoculation on the composition and structure of microbiota in different interaction zones,

such as the rhizosphere and endosphere, remain unclear. The genetics of a plant influence its ability

to attract, recognize, and interact with beneficial microbes while repelling harmful ones. Similarly,

microbial genetic traits determine their ability to colonize plant roots, promote growth, or provide

disease resistance. The plant genome includes specific genes that govern signaling and response

mechanisms activated upon microbial detection. These genes are tightly regulated and can trigger

various responses, from forming symbiotic associations and enhancing nutrient uptake to initiating

a robust immune response to prevent infection. Conversely, microbial genomes possess genes that

allow them to evade plant defenses or produce compounds beneficial for plant growth. Using

PGPR leads to a decrease in soil pH, enhancing the solubility of nutrients like phosphorus, iron,

zinc, manganese, and copper, thereby improving nutrient absorption by plants (Abd El-Aal and

Abd El-Rahman 2014). The use of 16S rRNA as a biomarker gene in genetic fingerprinting has

been crucial for identifying different isolates in various laboratories (Gomila et al. 2014). Many

Plants Growth-Promoting Rhizobacteria (PGPR) have been shown to produce various

phytohormones (Mareque et al. 2015). This research aimed to isolate new PGPR from the pine-

apple rhizosphere and comparing them to the known PGPR Acinetobacter baumannii OYA S31.

Additionally, maize (Zea mays) was used to study the effects of the obtained isolate on various

plant growth parameters in a greenhouse setting.

1.2 STATEMENT OF PROBLEM

3
Agricultural productivity faces growing challenges from various biotic and abiotic stresses, posing

significant risks to global food security. While traditional breeding methods have been useful, they

often fall short of meeting the increasing demands for higher crop yields and enhanced resilience.

Plant-microbe interactions are vital for plant health and productivity, affecting nutrient absorption,

disease resistance, and stress tolerance. However, the genetic mechanisms driving these

interactions are not well understood, limiting their potential use in strategies for crop improvement.

The limited understanding of the genetic basis of plant-microbe interactions impedes the

development of crops with enhanced traits for better yields, disease resistance, and adaptability to

environmental conditions. This research seeks to address this knowledge gap by exploring the

genetic factors that influence plant-microbe interactions, with the ultimate aim of improving crop

resilience and productivity. This statement emphasizes the current knowledge gap, the importance

of the research, and its potential contributions to advancing crop improvement efforts.

1.3 JUSTIFICATION OF STUDY

1. Global Food Security: With the world’s population on the rise, the need for increased food

production is becoming more urgent. To address this, it is essential to develop crops that are

not only high-yielding but also capable of withstanding various environmental stresses.

Gaining insight into the genetic basis of plant-microbe interactions presents a promising

approach to enhance crop health and productivity.

2. Biotic and Abiotic Stress Management: Crops face continuous challenges from various biotic

factors (such as pathogens and pests) and abiotic factors (like drought, salinity, and extreme

temperatures) that can significantly reduce yields. Plant-associated microbial communities

play a key role in mitigating these stresses by boosting disease resistance, enhancing nutrient

4
 absorption, and increasing stress tolerance. By decoding the genetic mechanisms underlying

these beneficial interactions, we can create crops with stronger natural defenses and greater

adaptability.

3. Sustainable Agriculture: The heavy dependence on chemical fertilizers and pesticides has

contributed to environmental degradation and biodiversity loss. Harnessing the power of

beneficial plant-microbe interactions provides an eco-friendly alternative, reducing the need

for chemical inputs while maintaining or even improving crop productivity. This research

could lead to more sustainable farming practices, aligning with global initiatives to combat

climate change and support ecological balance.

1.4 AIM AND OBJECTIVES OF THE STUDY

1.4.1 AIM OF THE STUDY

The Aim of this study is to explore the molecular and genetic mechanisms underlying plant-

microbe interactions, including how plants recognize and respond to microbes using Maize as a

model plant.

1.4.2 OBJECTIVES OF THE STUDY

The specific objectives of this study are to;

5
1. determine the plant genes that are involved in interactions with beneficial and harmful

microbes. This includes identifying genetic pathways related to disease resistance, symbiosis,

and nutrient acquisition.

2. explore how these key genes function and are regulated during plant-microbe interactions. This

involves studying gene expression patterns and the impact of genetic variations on these

interactions.

3. investigate how different microbes, including pathogens and beneficial symbionts, affect plant

genetic expression and overall plant health.

1.5 RESEARCH QUESTIONS

1. Which plant genes are critical for successful interactions with beneficial microbes?

2. How do plant genetic variations influence microbial interactions?

3. What are the molecular mechanisms underlying plant responses to microbial signals?

4. How do microbes influence plant gene expression and function?

5. What impact does the manipulation of plant-microbe interactions have on crop yield and

quality in field conditions?

6. How can knowledge of plant-microbe genetics contribute to sustainable agriculture pratices?

6
 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 BENEFICIAL PLANT-MICROBE INTERACTION

Plants host a remarkable variety of microbes in both their aboveground and belowground tissues,

collectively referred to as the plant microbiota. The genomes of these microbes, which live in close

association with plants, are commonly called the plant microbiome (Bulgarelli et al., 2013). Two

to five percent of rhizobacteria significantly influence plant development when reintroduced into

soil with competing microorganisms; these bacteria are known as PGPR. The plant's natural social

network is both dynamic and constant, as the plant does not exist in isolation. The surrounding

microorganisms continuously organize and sustain the colony (Kloepper et al., 1992). PGPRs are

rhizobacteria that support plant growth. In the rhizosphere, the soil layer closest to plant roots, a

group of beneficial bacteria resides. These bacteria form a cooperative relationship with plants,

providing various benefits that enhance plant development (Saravanan et al., 2011). Researchers

have extensively examined the beneficial effects of PGPR, a naturally occurring soil bacterium,

on plant health and productivity. These bacteria not only protect plants from pathogens and harsh

conditions but also enhance nutrient availability, stimulate plant growth, and strengthen root

development. Various mechanisms are involved in how PGPR benefits plants, including the

production of auxins, cytokinins, and gibberellins, which promote root and shoot growth (VanPeer

et al., 1989).

PGPR enhances plant resistance by activating defense systems and increasing tolerance to various

environmental factors such as drought, salinity, and extreme heat. Soil contains numerous types of

microorganisms, predominantly bacteria. The use of PGPR as bio-fertilizers and bio-pesticides has

7
gained significant attention in sustainable agricultural practices. Conventional agriculture's

frequent use of chemical pesticides and fertilizers can adversely affect human health, the

environment, and the sustainability of agricultural production. Consequently, sustainable and

environmentally friendly approaches are becoming increasingly crucial for enhancing plant growth

and agricultural productivity. Recently, PGPR has emerged as a promising solution for long-term

farming success (Gupta et al. 2015). PGPR significantly contributes to environmentally friendly

farming through various mechanisms. These bacteria promote plant development through both

direct and indirect methods.

2.2 DIRECT MECHANISM OF CROP IMPROVEMENT BY MICROBES

2.2.1 NUTRIENT ACQUISITION AND GROWTH PROMOTION

Limited nutrient availability and inadequate supply to plants severely impact their growth and

productivity. The availability of soil nutrients for plant uptake depends on various factors,

including soil composition, moisture content, texture, pH, and the existing microflora in the

rhizosphere (Vaishnav et al., 2016). Microbes have been found to assist plants in acquiring

nutrients through various mechanisms, including nitrogen fixation, increasing the surface area

accessed by roots, phosphate solubilization, and the production of HCN and siderophores (Pii et

al., 2015).

Nitrogen is a vital element in living systems, necessary for nucleic acid and protein synthesis. Soil

microbes play a crucial role by fixing atmospheric nitrogen, converting it into ammonia, and thus

making it available to plants through the nitrogen fixation process. Beneficial microorganisms,

particularly Plant Growth-Promoting Rhizobacteria (PGPR), have a significant impact on nitrogen

uptake in maize plants through the process of nitrogen fixation by making mitrogen accessible for

8
the pkant to take up, through the solubilization of nutrients improving plant's growth and

development.

The next essential macronutrient for plant growth and development is phosphorus. Although it is

abundant in agricultural soils, 30-65% of it exists in non-soluble organic forms (such as phosphates

and phosphodiesters) that plants cannot absorb (Khan et al., 2009). To meet plants' phosphorus

needs, fertilizers are often used. Additionally, phosphate-solubilizing bacteria from genera like

Pseudomonas, Alcaligenes, Bacillus, and Corynebacterium can convert these recalcitrant forms

into plant-accessible forms (Khan et al., 2009). These bacteria secrete organic acids or H+ ions,

which help solubilize inorganic phosphate that has formed complexes with calcium, aluminum,

and iron (Adesemoye and Kloepper, 2009). Similarly, the release of phytase by these bacteria

facilitates the conversion of organic phosphorus into its active, plant-available form. A diverse

range of beneficial microorganisms, including soil bacteria, fungi, and those associated with plant

roots, have the capacity to solubilize phosphorus in the soil, which would otherwise remain

insoluble. In a study by Researcher (Bechtaoui et al., 2020), a 25% reduction in the phosphorus

requirement was observed in phosphorus-solubilizing bacteria (PSB). Additionally, (Khan et al.,

2009) noted an enhanced effect of PSB when combined with other Plant Growth-Promoting

Rhizobacteria (PGPRs). Most bacteria capable of metabolite dissolution, typically through organic

acids, primarily rely on phosphorus for their synthesis in forms like ketone and gluconic acid

ketoforms. In these forms, the cations are chelated and bound to their respective hydroxyl and

carboxyl groups in phosphate. This facilitates their dissolution into the soil solution, making them

available for plant uptake (Heydari et al., 2007). Genera such as Arthrobacter, Bacillus,

Burkholderia, Enterobacter, Microbacterium, Pseudomonas, Rhizobium, Mesorhizobium,

9
Flavobacterium, and Serratia include PGPR bacteria capable of phosphorus dissolution (Riaz et

al., 2021)

Hydrogen Cyanide (HCN), a secondary metabolite produced by Gram-negative bacteria, is formed

through the oxidative reaction of glycine, catalyzed by HCN synthase. This flavoenzyme-mediated

reaction involves flavin adenine dinucleotide (FAD), while pyrrolnitrin inhibits the process.

Initially, microbial HCN production was considered a plant protective mechanism, but it was later

found to enhance phosphorus availability for plants (Rijavec and Lapanje, 2016). Iron, an essential

micronutrient for plant growth and development, is abundant in the earth's crust but often scarce

in its bioavailable form in most soils, typically existing as ferrous (Fe2+) iron. Since plants require

the ferric (Fe3+) form, bacteria secrete siderophores—iron-binding ligands—to convert Fe3+ for

their use. Siderophores are low molecular weight, water-soluble compounds of microbial origin

that sequester iron (Fe3+) and transport it into bacterial cells (e.g., ferripyoverdine, a combination

of Fe3+ and pyoverdin) for use in nitrogen fixation, respiration, and photosynthesis (Saha et al.,

2016).

2.2.2 PRODUCTION OF SIDEROPHORE AND OSMOTIC BALANCE

Siderophores are compounds that bind to iron, increasing its availability for plants (Goswami et

al., 2016). Siderophore production by microbes and pH alterations occurring in the rhizosphere

meet the nutritional requirements of plants. In conditions of low iron availability, bacteria produce

siderophores, which are small chemical compounds, to increase their capacity for iron absorption.

Iron is crucial for all photosynthesis-dependent organisms because it serves as a crucial enzyme

cofactor in various metabolic processes, including photosynthesis, amino acid synthesis, oxygen

transfer, nitrogen fixation, and respiration (Tian et al., 2009). Iron is a common chemical element

10
on Earth, typically occurring in two oxidative states: Fe2+ and Fe3+. Plants have restricted access

to the latter state because of the formation of insoluble iron oxides and hydroxides (Khoshru et al.,

2020). Studies suggest that certain plant growth-promoting bacteria (PGPB) produce small

molecules (with molecular weights ranging from 400 to 1500 Da) capable of extracting iron from

the soil (Shanmugaiah et al., 2015). The buildup of salt ions frequently disrupts the osmotic

equilibrium in plants. PGPR enhances the plant-water relationship by increasing the production of

osmolytes that are taken up by plants at the root surface (Mishra et al., 2018). This stimulation

promotes the uptake and accumulation of compatible solutes (osmolytes) like proline, glycine,

polyamines, etc., which are crucial for preserving osmotic balance and preventing oxidative

damage to cellular components (Sandhya et al., 2010). These compatible solutes influence

hydraulic conductivity, thereby regulating water potential and stomatal opening.

2.2.3 PHYTOHORMONE PRODUCTION

PGPR-mediated exogenous release of hormones, metabolites, and enzymes modulates plant

hormonal balance, thereby enhancing their growth under extreme stress conditions. These

microbes can alter water and nutrient uptake, plant morphology, metabolism, and tolerance to

various environmental stresses, significantly contributing to plant growth and development (Fahad

et al., 2015). Indole-3-acetic acid (IAA) production, a common trait of PGPR, improves the

resilience of plants under stress. PGPR uses tryptophan from root exudates to induce IAA synthesis

in the rhizosphere (Spaepen et al., 2007). PGPR-produced auxin triggers changes in the expression

of genes related to the cell wall, hormones, and defense mechanisms, resulting in reduced stomatal

size and density, increased root biomass, and the activation of auxin response genes that are crucial

for plant growth and development (Llorente et al., 2016). PGPR-mediated production of

gibberellins enhances hypocotyl and stem growth, thereby aiding in the regulation of leaf and root

11
meristem size (Martínez et al., 2016). Cytokinins, a group of purine-based plant hormones, are

commonly associated with regulating cell division and differentiation in meristematic tissues,

chloroplast maturation, and stomatal conductance (Cassán et al., 2014). While cytokinin

production is a common trait in PGPR, it affects the endogenous cytokinin pool by inducing its

synthesis or altering its homeostasis in plants (Kapoor and Kaur, 2016). Since the auxin-to-

cytokinin ratio is crucial for determining cell fate, inoculating cytokinin-producing B. subtilis in

drought-stressed lettuce plants promotes growth by modulating root-to-shoot signaling (Arkhipova

et al., 2007).

Beneficial microbes can enhance plant growth by influencing the hormonal balance within plants.

This positive effect can occur through the secretion of microbial small secondary metabolites (SM)

that act as hormone-like plant growth regulators, or through the production of SM and proteins

that allow microbes to modulate plant defense hormone signaling, thereby facilitating successful

colonization of plant tissues (Stringlis et al., 2018). Many microbial species associated with plants,

including bacteria and fungi, produce indole-3-acetic acid (IAA) or auxin-mimicking molecules,

which directly influence plant growth and development (Garnica-Vergara et al., 2016).

Additionally, other microbial phytohormones or phytohormone-like molecules, such as cytokinins,

gibberellins, and analogues of defense-related hormones like salicylic acid (SA) and jasmonic acid

(JA)-isoleucine, are primarily produced to aid microbial colonization by modulating plant

immunity (Stringlis et al., 2018).

2.3 INDIRECT MECHANISM OF CROP IMPROVEMENT BY MICROBES

12
PGPR indirectly protect plants from or mitigate the effects of phytopathogens by producing

suppressive chemicals that enhance the plant's innate resistance. In this process, PGPR generate

hydrolytic enzymes (such as chitinases and cellulases), antibiotics in response to plant pathogens

or diseases, and protect the entire plant system from various pathogens and pests. Additionally,

they produce volatile organic compounds (VOCs) and exudates via photosynthesis (Hasan et al.,

2024).

2.3.1 STRESS MITIGATION

Stress refers to any factor that hinders plant development. One of the primary challenges to

sustainable agricultural productivity is the variety of stresses caused by the soil environment on

plant growth, which can be categorized into biotic and abiotic groups. Abiotic stress is the leading

cause of over 30% of global crop losses. Drought or aridity stress, resulting from dryness, salinity,

and high temperatures, is the most prevalent type of abiotic stress that impedes plant growth and

productivity. Researchers have extensively studied the role of PGPR in protecting plants from

environmental stresses, using microorganism strains like Pseudomonas putida and Pseudomonas

fluorescens. These strains significantly mitigate water salinity and other abiotic stresses and can

remove cadmium ions from the soil (Baharlouei et al., 2011). The selection pressures affecting

plant growth and development are divided into biotic factors (such as bacteria, viruses, and grazing

by higher animals) and abiotic factors (such as heavy metals, extreme temperatures, drought, and

salinity) (Bhat et al., 2020a; Bhat et al., 2020b) Abiotic stresses significantly impact various

aspects of plant growth, posing a serious threat to plant survival (Nadeem et al., 2019) These

stresses lead to a notable decrease in germination rates and severely affect photosynthetic activity,

carbon assimilation, and overall crop productivity (Chowdhury et al , 2016). In such challenging

13
conditions, the phytomicrobiome plays a crucial role in helping plants, in association with

microbes, survive extreme environmental conditions (Cappelli et al., 2022; Sokol et al., 2022).

2.3.2 ANTIBIOTIC PRODUCTION

Microbial antagonists can substitute traditional pesticides to manage plant diseases in crops. PGPR,

along with Bacillus spp. and Pseudomonas sp., play a key role in preventing the spread of harmful

bacteria by producing antibiotics. Over the past 20 years, one of the most promising fields in plant

sciences has been the production of antibiotics by PGPR to combat numerous plant pathogens,

with extensive research into biocontrol mechanisms (Ulloa-Ogaz et al., 2015). Most Pseudomonas

species produce antibiotics such as oomycin A, cepaciamide A, ecomycins, and viscosin, as well

as pyrrolnitrin, pyoluteorin, 2,4-diacetylphloroglucinol, rhamnolipids, and pyoluteorin. Bacillus

species generate several lipopeptide antibiotics like surfactin and bacillomycin, along with other

antibiotics and antifungal agents. Bactericides are further classified into volatile and non-volatile

substances. Non-volatile antibiotics include polyketides, cyclic lipopeptides, aminopolyols, and

heterocyclic nitrogenous compounds. Examples of volatile antibiotics are alcohols, aldehydes,

ketones, hydrogen cyanide, and others (Fouzia et al., 2015).

2.3.3 HYDROLYTIC ENZYMES PRODUCTION

PGPR produces protective enzymes, functioning as a biopesticide. By suppressing

phytopathogenic agents, PGPR promotes plant growth and generates compounds with antibiotic

and antifungal properties that act as defense mechanisms. During this process, hydrolytic enzymes

like chitinase and glucanase are produced. Bacteria that produce chitinases and beta-glucanases

inhibit fungal growth since these enzymes target the main components of fungal cell walls.

14
Sinorhizobium fredii KCC5 and Pseudomonas fluorescens LPK2, for instance, produce chitinase

and beta-glucanase, preventing Fusarium udum from causing plant wilt (Kumar et al., 2010).

2.4 ROOT EXUDATES AND SIGNALLING

Throughout a plant's life, its roots engage in beneficial interactions with soil-dwelling microbes

while simultaneously managing infections from pathogenic microorganisms. Soilborne pathogens

can persist in the soil for varying durations, either as saprophytes on plant debris and organic matter

or in dormant forms such as sclerotia, chlamydospores, oospores, and melanized mycelia. These

pathogens are activated by root exudates (De Coninck et al., 2015). For instance, phenolic acids,

sugars, and free amino acids in watermelon root exudates significantly enhance the spore

germination and sporulation of Fusarium oxysporum f. sp. niveum (Hao et al., 2010). Similarly,

tomato root exudates promote the germination of microconidia of the tomato pathogens Fusarium

oxysporum f. sp. lycopersici and Fusarium oxysporum f. sp. radicis-lycopersici, with the

stimulation level varying with plant age (Steinkellner et al., 2005). Additionally, fungal pathogens

can detect root exudates, guiding their hyphal growth towards the roots. The chemotropic response

of Fusarium oxysporum to tomato roots involves the activity of root-secreted class III peroxidases

(Turrà et al., 2015). Under favorable conditions, soilborne pathogens invade plants through the

root system, typically affecting roots and other underground parts, though symptoms often appear

on above-ground parts (Koike et al., 2003). Infected plants may exhibit root rots, inhibited root

development, stunted growth, seedling damping-off, stem and collar rots, wilting, or even death

(De Coninck et al., 2015; Katan, 2017).

The plant-associated microbiota, which includes bacteria, fungi, and other microorganisms, exists

within complex communities and exhibits a range of beneficial traits. These include the

15
suppression of plant pathogens and the transformation and translocation of essential nutrients in

the soil, facilitating their uptake by plants. These interactions promote plant growth and enhance

productivity (Upadhyay et al., 2022). Plant root exudation of signaling molecules, such as

jasmonic acid and salicylic acid, regulates the initial stages of microbial colonization of plant root

(Doornbos et al., 2011). Root exudates play a crucial role in signaling within the rhizosphere,

facilitating inter-organismal communication under varying conditions (Figure 2.1). The molecules

secreted by roots support microbial growth and activity in the rhizosphere. In return, these

microbes aid plants in acquiring soil resources, protect against pathogens, and promote growth by

increasing root hair proliferation, branching, and leaf surface area. They also elevate indigenous

hormone levels and enhance carbohydrate accumulation, thereby improving plant vigor and

biomass from germination to full maturity and even into senescence (Vocciante et al., 2022).

16
Plate 2.1: Plant-microbiome interactions: (a) PGPR association with roots of the plant, (b)

Microbial interactions enhancing cellular signaling, and (c) Signaling pathway along different

modules affecting gene expression and, as such, overall performance of the plant.

17
2.5 PLANT-MICROBE MOLECULAR COMPLEX

In highly diverse ecosystems, numerous plant species coexist with a variety of microorganisms,

forming relationships that can be helpful, harmful, or neutral. These interactions enable plants to

connect with other microorganisms, which can either enhance or hinder their survival. Over the

past several decades, scientists have explored the reasons and mechanisms behind the varying

interactions between plants and microorganisms in their environments. One key factor is their

genotype, which contains the instructions for adaptation and survival (Jones and Dangl, 2006).

Ecosystems rely on the cooperation between plants and microorganisms, and this connection is

based on intrinsic molecular interactions. For instance, through photosynthesis, plants convert

sunlight into chemical energy, producing oxygen and essential organic compounds like glucose.

These biomolecules support the growth of various microorganisms. On land, bacteria and fungi

break down organic matter using specific enzymes, releasing nutrients in simpler forms that plants

can absorb (Hwang et al., 2017). However, some plant-microorganism relationships involve other

molecular dynamics. An example is nitrogen fixation, where symbiotic bacteria in the root nodules

of legumes convert atmospheric nitrogen into ammonia using nitrogenase enzymes. Mycorrhizal

fungi enhance the nutrient absorption capacity of many plants, particularly phosphorus, by

extending the root absorption area with their hyphal networks. These interactions are essential for

the nutrient cycle and maintaining ecosystem balance (Hwang et al., 2017; Zipfel and Oldroyd,

2017). Since the 1980s, significant discoveries have been made about the complex interactions

between plants and their microbiota. These findings reveal highly coordinated molecular networks

that facilitate diverse biological interactions (Zipfel and Oldroyd, 2017). For example, plant cells

have specialized structures that enable them to detect microbial characteristics. By recognizing

these motifs, cells can activate defenses or establish symbiotic metabolic pathways depending on

18
the specific microbes (Hwang et al., 2017; Zipfel and Oldroyd, 2017). These microorganisms also

have the ability to synthesize RNA and translate peptide-mimicking molecules. These processes

trigger reactions in plant cells, creating an environment conducive to harmonious coexistence and

providing an adaptive advantage to microbes. Specific plant structures, such as root nodules and

symbiotic relationships with mycorrhizal fungi, act as catalytic agents at the metabolic level and

are considered key epicenters in organismal relationships, ranging from cooperative collaboration

to biological antagonism (Zipfel and Oldroyd, 2017).

When studying the complex interactions between beneficial microorganisms and their

environment, researchers encounter a diverse and constantly changing landscape that necessitates

a detailed examination of regulatory processes in controlled settings (Compant et al., 2010).

During plant-microbe interactions, a mutual recognition system is crucial for effective physical

and molecular coordination. This system involves Nod and Nif genes, microbial communication

molecules, auxins and cytokinins, resistance genes (R genes), elicitors, and pathogen-associated

molecular patterns (PAMPs). These interactions, influenced by the unique genetics of each plant

and their cellular adaptations, result in regulated signal exchanges. Consequently, molecular

connections ensure optimal functional integration in the shared environment, reflecting a

coevolution refined over many eras (De Vos et al., 2005; Hacquard et al., 2017). Recent advances

in genomics have shown that plants have adapted to a broad spectrum of biotic interactions,

extending beyond their relationships with parasitic and herbivorous organisms to include

beneficial symbionts. The signaling pathways plants use to manage these interactions often share

common features, indicating that their regulatory structures have evolved to both defend against

aggressors and enhance symbiotic associations. Studies on this phenomenon provide a deeper

understanding of how plants respond to environmental factors and their resulting adaptability

19
(Reymond et al., 2004; Morris et al., 2007). Plants and microorganisms like bacteria, fungi, and

viruses communicate and respond to each other through a complex network of molecular pathways,

referred to as plant-microbe interactions.

2.5.1 RECOGNITION AND SIGNAL TRANSMISSION

Plant as sessile organisms constantly face microbial attacks. To combat this, plants are equipped

with pattern recognition receptors (PRRs) that identify conserved molecular patterns associated

with microbes, known as pathogen-associated molecular patterns (PAMPs). This recognition

triggers a cascade of signaling events. Elicitors, which are vital for infection survival, are

conserved structures recognized by the innate immune systems of plants as either microbe-

associated molecular patterns (MAMPs) or PAMPs, using PRRs. These PRRs, located on the plant

surface, detect general elicitors such as fungal chitin, β-glucans from oomycetes, elongation factor

Tu (EF-Tu), peptidoglycan (PGN), lipopolysaccharides (LPS), Ax21 (an activator of XA21-

mediated immunity in rice), and flagellin (Newman et al., 2013). Despite the extensive and diverse

sets of receptor-like proteins (RLPs) and receptor-like kinases (RLKs) present in various plant

species, only a few have been functionally elucidated. Different plant species seem capable of

detecting a wide range of MAMPs (Ranf, 2017). When confronted with pathogen effectors—

proteins secreted by microbes to manipulate host cells—plants can trigger effector-triggered

immunity (ETI), a faster and more robust defense mechanism involving the specific recognition

of effectors by plant resistance (R) proteins.

2.5.2 ENVIRONMENTAL EFFECTS AND MICROBIAL SIGNALS IN PLANT

DEFENSE

20
Environmental factors like temperature and light significantly impact the immune system of plants

(Hua, 2013). This indicates that plants integrate information from both microbial and

environmental sources to adapt their physiological responses. Recent research has focused on

analyzing transcriptional changes in the Arabidopsis resistance protein RRS1-R, which provides

protection against Ralstonia solanacearum infection (Deslandes et al., 2002). This protein,

potentially resulting from gene fusion, features a TIR-NBS-LRR domain (proteins with an amino-

terminal domain similar to that of Toll and interleukin-1 receptors) common in plant resistance

proteins, and a WRKY domain (a 60-amino acid region with the conserved sequence WRKYGQK

at its N-terminus and a distinctive zinc finger-like motif) at its carboxyl-terminal end, typical of

plant transcriptional regulators. The process of integrating signals from plant gene regulation is

still a subject of active research, with many aspects yet to be explored. A key mechanism in this

integration involves a versatile transcription factor (TF), which is influenced by various signaling

pathways. This can occur either at the gene promoter, where different TFs interact with multiple

cis-regulatory elements (CREs), or at the CREs themselves, which are linked to stress-responsive

genes. Despite the identification of several CREs, accurately predicting gene expression in

response to stress remains difficult (Wick et al., 2003).

Microbes frequently generate multiple MAMPs, allowing plants to adjust the PTI signaling

network by detecting various combinations of these molecules, which leads to a context-specific

immune response (Vlot et al., 2009). It is commonly believed that PAD4 boosts SA signaling.

However, new evidence indicates that SA might primarily activate PAD4 rather than directly

strengthen the defense against biotrophic and hemibiotrophic pathogens. This challenges the view

that PAD4 has a more crucial role in bacterial defense than SA. Therefore, it is essential to study

PAD4 and SA both separately and together to better understand their roles and interactions in plant

21
defense. (Sharifi et al., 2018) proposed that host plants produce specific volatile compounds when

attacked by antagonistic microbes. These secreted metabolites differ based on the plant's resistance

to the invading microorganisms. Resistant plant varieties often release various chemical

compounds, including limonene and linalool, while nonanal acts as a molecular marker.

Additionally, exposure to exudates from resistant plants boosts the defense mechanisms of

susceptible varieties and reduces fungal diseases. Similarly, beneficial microbes induce the release

of volatiles to protect against insect damage. For example, plants treated with plant growth-

promoting (PGP) rhizobacteria metabolize compounds like indole and beta-caryophyllene,

enhancing resistance to antagonists. Likewise, symbiotic fungi (endophytes and mycorrhizal

associations) enhance the production of volatiles in their host plants to guard against insect threats.

This process is depicted in Figure 2.2. Beneficial microbes similarly trigger the release of volatiles,

offering protection against insect herbivory. The application of growth-promoting rhizobacteria

prompts plants to produce defensive volatiles like indole and beta-caryophyllene. Additionally,

symbiotic relationships with fungi, such as endophytes or those forming mycorrhizal connections,

affect these volatile patterns, providing a defense mechanism against insect attackers. JA stands

for jasmonic acid; SA for salicylic acid; PAMP for M/pathogen-associated molecular pattern;

MeSA for methyl salicylate. Adapted from (Sharifi et al., 2018).

22
Plate 2.2: Microbial pathogens impact host plants, prompting them to emit volatile compounds.

(Sharifi et al., 2018).

23
2.5.3 CHEMOTAXIS AND QUORUM SENSING

While the mechanics of bacterial chemotaxis have been fully elucidated, their underlying

mechanisms remain unclear. Conventionally, chemotaxis has been viewed as a bacterial strategy

for food searching and optimizing nutrient uptake. However, some compounds with low nutritional

value act as chemoattractant, while highly nutritious compounds do not. Chemotaxis also serves

broader ecological roles, such as guiding colony expansion, identifying hosts or symbiotic partners,

and promoting microbial diversity through spatial segregation in communities. Metabolic coupling

between microbial communities offers benefits like enhanced motility and chemotaxis, helping to

address the challenges of small organism size. Chemotaxis is crucial for forming microbial

communities in various habitats. For instance, nitrogen-fixing filamentous cyanobacteria like

Anabaena spp. release signals at the edges of their heterocyst that attract Pseudomonas spp.,

improving nitrogen fixation (Paerl and Gallucci, 1985). Similarly, sulfate-reducing bacteria

Desulfonema spp. exhibit sliding mobility to reach the mucus layer of Thioploca spp., promoting

sulfate reduction and reoxidation between the two species (Fukui et al., 1999). Microorganism

chemotaxis and motility are fundamental to symbiotic interactions. For instance, dental plaque

consists of at least 12 different microbial taxa organized into consortia (Mark Welch et al., 2016).

This precise organization is crucial for the function and durability of dental plaques in the oral

environment. The microorganisms in these samples are believed to exhibit motility, possibly

through flagella or type IV pili, and primarily act as episymbionts, forming symbiotic relationships

on the surfaces of other microorganisms (Castelle et al., 2018). These examples highlight the

importance of motility and chemotaxis as essential tools for microbes to identify and form specific

symbiotic relationships, directly influencing their survival and success in the ecological realm.

24
Quorum sensing (QS) is a biological process where bacteria communicate using signaling

molecules, monitoring population density and adjusting gene expression (Daniels et al., 2004). QS

regulates various processes such as bioluminescence, sporulation, competence, antibiotic synthesis,

biofilm formation, and secretion of virulence factors (Williams and Cámara, 2009). Bacterial

communication extends to interactions with plants (Williams and Cámara, 2009), where molecules

like N-acyl-L-homoserine lactones (AHLs) influence behavior depending on the bacterial strain

(Ortíz-Castro et al., 2009). AHLs are prevalent in pathogenic Gram-negative bacteria such as

Pseudomonas aeruginosa and Rhizobium radiobacter, as well as in plant growth-promoting

bacteria like Burkholderia graminis (Fuqua et al., 2001). These molecules are pivotal in microbial

functions including symbiosis, virulence, and antibiotic production. Plants can detect bacterial

AHLs affecting gene expression in specific tissues, influencing development and triggering

defense responses (Liu et al., 2022).

2.5.4 PHYTOHORMONE SIGNALING

Salicylic Acid (SA), a beta hydroxy phenolic acid, is synthesized extensively in prokaryotes and

plants. It acts as a critical signaling molecule in plant defense mechanisms against biotrophic

pathogens, which depend on living plant tissue. SA induces the expression of defense genes and

initiates systemic acquired resistance (SAR). It serves as a pivotal hormone in plant innate

immunity, providing resistance in local and systemic tissues during biotic attacks, hypersensitive

responses, and cell death (Ding and Ding, 2020). Recent research has focused on identifying NPR3,

NPR4, and NPR1 as potential SA receptors, alongside α-ketoglutarate dehydrogenase (KGDHE2),

various glutathione S transferases (GSTF), and SA binding proteins. Apart from their role in

pathogen defense signaling, some of these SA binding proteins may also participate directly or

indirectly in other plant processes (Kumar, 2014).

25
In recent years, the phytohormone jasmonic acid (JA), its amino acid conjugates like JA-Ile, and

related derivatives have emerged as crucial signaling molecules involved in plant growth and stress

resilience. These hormones play a significant role in defending against diseases caused by

necrotrophic pathogens that induce host cell death (Ghorbel et al., 2021). They regulate the

expression of genes associated with defensive responses.

26
Table 2.1: Microbial-mediated interactions with host plants (Jain et al., 2024)

27
2.5.5 SECONDARY METABOLITE PRODUCTION

Phytoalexins are antimicrobial metabolites with low molecular weight that plants produce in

response to infection (Kuć and Rush, 1985). These lipophilic compounds, a product of the

secondary metabolism in plants, often accumulate at infection sites in quantities sufficient to

inhibit bacterial and fungal growth. According to (Hasegawa et al., 2010), the absence of early

phytoalexin accumulation in susceptible rice allows fungal growth, whereas in resistant rice, rapid

phytoalexin production associated with a hypersensitive response significantly restricts fungal

development.

2.6 GENOMIC AND PROTEOMICS APPROACH FOR PLANT-MICROBE

INTERACTION.

The incorporation of genetic innovations offers the potential to enhance PGPR-based technologies,

promoting efficacy, sustainability, and environmental compatibility in addressing plant stresses.

In the field of nanotechnology, developed countries like the United States, China, and Japan

strategically employ sophisticated approaches to significantly boost agricultural productivity (Rani

et al., 2020). This strategic implementation involves a multifaceted exploration of cutting-edge

techniques, encapsulating a variety of approaches aimed at optimizing key aspects of agricultural

processes. Within the United States, nanotechnology applications in agriculture span various

domains. Environmental genomic research is currently among the most effective methods for

acquiring extensive knowledge about evolutionary history, as well as structural, functional, and

ecological biodiversity. "Metagenomics" refers to the analysis of all genetic material from

microbes directly obtained from relevant environmental samples. This approach is frequently used

to study the complex microbial communities found in environmental materials through nucleotide

28
sequence analysis. The two main methodologies in metagenomic research are targeted sequencing

and shotgun sequencing, with the choice between them depending largely on the specific type of

environmental study being conducted (Hugenholtz et al., 2008).

Nanosensors have a crucial role in monitoring soil conditions, offering real-time data for accurate

irrigation and fertilization strategies. Moreover, nanomaterials aid in developing controlled release

systems for pesticides, reducing environmental impact while optimizing effectiveness. China

incorporates nanotechnology to tackle soil fertility and crop protection challenges. Recent

developments in omics methodologies provide a promising pathway to tackle the issues impacting

crop yield, by uncovering the advantages of plant-microbe relationships (Olanrewaju et al., 2024).

Advances in analytical techniques have significantly enhanced our understanding of the complex

dynamics involved in these relationships (Sharma et al., 2024). Omics technologies such as

genomics, transcriptomics, and metabolomics have been crucial in investigating the biochemical,

physiological, and molecular dimensions of plant-microbe interactions under diverse

environmental conditions (Tiwari et al., 2024). The complex interplay between plants and

microbes is crucial for ecosystem dynamics and sustainable agriculture, shaped by host-specific

factors, soil conditions, environmental changes, and human activities (Rane et al., 2022). Grasping

these elements is pivotal in deciphering the intricate nature of symbiotic relationships. Omics

methodologies such as genomics, transcriptomics, proteomics, and metabolomics shed light on the

molecular complexities underlying plant-microbe symbiosis (Sarim et al., 2020).

Genomics identifies critical genes, transcriptomics unveils the dynamics of gene expression,

proteomics pinpoints essential proteins, and metabolomics profiles small molecules, providing a

holistic view. Multi-omics approaches allow for the exploration of diverse interactions,

encompassing obligatory symbioses like those between arbuscular mycorrhizal fungi and plants,

29
and interactions with non-mycorrhizal microbes such as biofilms (Mishra et al., 2022). The

molecular signaling mechanisms in microbial interactions with host plants are intricate and

essential for symbiosis, pathogenesis, and adaptation to the environment. Microbes like Rhizobium

leguminosarum produce phytohormones such as auxins and cytokinins, which activate plant

receptors to stimulate growth and enhance nutrient uptake (Meena et al., 2023). Microbes employ

diverse strategies, such as producing exopolysaccharides (EPS), binding through pili/fimbriae,

secreting adhesins, and enzymatically breaking down plant cell wall components (Dutta et al.,

2023; Niazi et al., 2023). Furthermore, chemical gradients like root exudates direct microbial

migration and colonization on plant surfaces (Dhiman et al., 2024). The secretion of molecular

effectors by microbial pathogens involves advanced mechanisms to manipulate host cellular

processes. Pathogens utilize various secretion systems, including T3SS, T4SS, T6SS, outer

membrane vesicles (OMVs), and direct secretion systems, to deliver effector proteins into host

cells (Maphosa et al., 2023; Wangthaisong et al., 2023).

Genomic, transcriptomic, proteomic, and metabolomic technologies have revolutionized

agriculture, medicine, food science, and life science. Recent findings from plant-microbe

interaction omics provide deep insights into the complex interactions between plants and microbes,

highlighting their impact on crop productivity, sustainability, and environmental resilience

(Sindelar, 2024). These advances have uncovered a diverse array of plant-associated microbial

species and their roles in nutrient cycling, disease control, stress tolerance, and plant-microbe

signaling networks. Omics data has identified microbial traits that enhance plant growth, stress

resistance, and nutrient uptake. Leveraging these insights, omics-driven strategies have been

developed to harness beneficial plant-microbe symbioses, including biofertilization, pest

management, and improving plant health and productivity. These approaches aim to enhance soil

30
fertility, reduce chemical dependency, and promote agricultural sustainability (Ramlal et al., 2023).

Precision agriculture utilizes plant-microbe interaction omics to optimize crop performance across

various environments by fine-tuning microbial communities. Combined with advanced

computational tools, omics data enables predictive modeling, microbial consortia design, and

agricultural management, thereby increasing productivity and resilience (Shoaib et al., 2023).

Studies have indicated that plant-associated microbes, including those found in the diet, can

influence the human microbiome, immune function, metabolism, and susceptibility to diseases.

Omics-based approaches help uncover the molecular mechanisms behind the interactions between

plant-associated microbes and the human microbiome (Bashiardes et al., 2016). By analyzing the

genomic, transcriptomic, proteomic, and metabolomic profiles of plants and human-associated

microbes, researchers have identified microbial-derived metabolites, proteins, and signaling

molecules that affect host physiology and immune responses (Saravanakumar et al., 2022). Some

plant-associated microbes produce bioactive metabolites that can kill human pathogens and

support the growth of commensal bacteria (Kandasamy and Kathirvel, 2023). Omics-based

screening methods can identify these microbial-derived compounds and uncover their mechanisms

of action, paving the way for new antimicrobials or probiotics to treat infectious diseases,

inflammatory disorders, and metabolic syndromes (Garia et al., 2024). Additionally, plant-

microbe interaction omics can aid in personalized medicine by highlighting individual variations

in microbial communities and their interactions with dietary components (Wright et al., 2023;

Speckmann et al., 2024).

The role of plant-associated microbial communities in influencing the health and growth of the

host is becoming increasingly recognized. Therefore, gaining a genomic-level understanding of

plant-microbe relationships could enhance agricultural productivity with the help of microbes.

31
Numerous studies have emphasized the importance of accurately characterizing plant microbiomes,

with a greater emphasis on microbial diversity rather than gene function.

2.7 TRANSCRIPTION FACTORS IN PLANT–MICROBE INTERACTIONS

Transcription factors (TFs) are pivotal in regulating key biological processes in plants. They

interact either directly with cis-type elements on gene promoters or through protein interactions.

It is crucial to maintain optimal expression levels of essential genes involved in biosynthesis and

development for proper plant growth and survival. TFs serve as central regulators of gene

transcription, influencing a variety of genomic sequences. They play a critical role in modulating

plant physiology, particularly in response to biotic and abiotic stresses, and are essential for

regulating secondary metabolism (Chowdhary et al., 2023). When a host detects a pathogen, it

triggers the activation and amplification of specific enzymes, hormones, and metabolites crucial

for its response. Gene regulation is fundamental in the interaction between host and pathogen

during the immune response. Numerous studies have identified genes encoding key proteins

involved in plant-pathogen interactions, many of which directly or indirectly contribute to the

host's ability to recognize pathogens. Most research to date has focused on genomic and

transcriptomic findings, emphasizing transcription factors (TFs) and regulatory elements that

significantly influence the remodeling of the host cell's transcriptomic profile. This remodeling

enhances the production of defense molecules. Interestingly, initial stages of these defense

responses often involve the expression of various stress-response genes (such as WRKY TFs) and

enzymes that scavenge reactive oxygen species (ROS). These TFs typically feature a WRKYGQK

sequence domain and a characteristic zinc-finger motif (Eulgem et al., 2000).

32
(Peng et al., 2012) investigated the function of the OsWRKY30 gene in the defensive responses

of rice (Oryza sativa) under various conditions. These included exposure to JA (jasmonic acid),

its derivative methyl jasmonate, SA (salicylic acid), and two known rice pathogenic fungi:

Rhizoctonia solani and Magnaporthe grisea. Following exposure to JA and SA, there was a

notable increase in OsWRKY30 transcript levels. Additionally, the expression of this gene was

positively associated with the activation of several defense-related genes, particularly those

involved in JA synthesis and it signaling pathways, such as lipoxygenase (LOX), allene oxide

synthase 2 (AOS2), PR-3, and PR-10. Moreover, heightened expression of OsWRKY30 resulted

in elevated JA levels in response to infection by the aforementioned pathogenic fungi.

RNA also plays a critical role in interactions between oomycetes and their host plants. For instance,

Phytophthora employs the effector PSR1 to target specific components of the RNA pathway in

plants, altering immunity and facilitating infection (Gui et al., 2022). Notably, the WY domain of

PSR1 is crucial for initiating infection and countering RNA silencing in numerous plant species

(Zhang et al., 2019). Recent research has explored the relationship between miRNAs and target

genes that confer resistance to Verticillium wilt in cotton. This investigation involved constructing

small RNA (sRNA) libraries and genomic sequencing, which identified 383 miRNAs. Among

these, GhmiR165 and GhmiR395 were identified as pivotal in response to Verticillium dahliae

infection. GhmiR165 is involved in vascular development and secondary cell wall formation via

the GhmiR165-REV pathway, while GhmiR395 regulates sulfur assimilation through the

GhmiR395–APS1/3 axis (Mei et al., 2022). Additionally, the interaction between GhmiR477 and

CBP60A molecules was found to influence cotton's response during advanced infection stages.

GhmiR477 targets and degrades GhCBP60A mRNA post-transcriptionally, with inhibiting

GhmiR477 reducing resistance and silencing GhCBP60A enhancing it (Hu et al., 2022).

33
34
 CHAPTER THREE

3.4. MATERIALS AND METHODS

3.5.AREA OF STUDY

Rhizospheric soil samples were collected from an unaltered, unpolluted field in phase three of the

Federal University Oye-Ekiti in Ekiti state, Nigeria, located at coordinates 7°46’36.1”N latitude

5°18’43.5”E longitude. Oye-Ekiti, part of this state, provides a unique mix of urban development

and rural surroundings, creating a dynamic setting for this research.

3.6.SOIL SAMPLE COLLECTION

Rhizospheric soil for bacterial isolation was collected from a depth of 15–20 cm around the roots

of a pine-apple plant (Ananas comosus) in Phase 3, Federal University, Oye-Ekiti, Ekiti state,

Nigeria, using a hand trowel and a sterilized shovel. This location is at approximately 7°46’36.1”N

latitude 5°18’43.5”E longitude, with annual temperatures ranging from a high of 38°C to a low of

25°C. The soil was removed from the plants by vigorously shaking them by hand for 10 minutes

before collecting the rhizospheric soil from the roots of the Ananas comosus (Pine-apple) plants.

Ten grams of rhizospheric soil samples were placed in sterilized universal bottles, stored in an

icebox at 4°C, and transported to the microbiology laboratory run by Professor Olubukola

Oyawoye. There, the samples were sorted, tagged, and divided into nine batches labeled A to I

(Waday et al., 2022).

35
Map 1: Map showing the area of study

Map 2: Map showing where samples are collected.

36
3.6.1. SOIL SAMPLING FOR METAGENOMICS ANALYSIS

The soil sampling tools, including universal containers, beakers, hand trowels, and sample bags,

were disinfected by immersing them in 70% ethanol for 10 minutes and allowing them to air dry.

Using a clean hand trowel and disinfectant, homogenized soil samples were placed into fresh

beakers. Ten grams of soil samples were measured with a weighing balance and then placed into

sterile universal containers labeled A to I, which were kept inside a clean sampling bag. The bags

were clearly labeled with batch numbers, dates, and other pertinent information, and then stored

in a deep freezer. The same sampling procedure was repeated to obtain samples for bacterial

isolation and characterization.

3.6.2. SOIL SAMPLE ANALYSIS

Soil samples were taken from sorted bags and measured using a weighing balance. Sixty grams of

soil were transferred into universal containers, and duplicates were made. The samples were sealed

in sterile polyethylene bags and sent to the Faculty of Agriculture at Federal University Oye-Ekiti,

Ikole campus, Ekiti state, for soil analysis. The parameters measured included moisture content,

pH, electrical conductivity, organic carbon, organic matter, available nitrogen, phosphorus,

potassium, sodium, calcium, magnesium, and total sulfate.

3.7.ISOLATION AND CFU DETERMINATION OF BACTERIAL STRAINS

Isolation of the organism was done through the serial dilution technique (Emami et al., 2020). One

gram of soil samples was mixed with 9 ml of distilled water in a beaker and stirred for 10 seconds

to achieve homogeneity. From this mixture, 1 ml was transferred to a 9 ml blank diluent to obtain

a 10-1 dilution. This suspension was mixed, and 1 ml of the diluted mixture was then transferred

to another 9 ml blank to achieve a 10-2 dilution. This process was continued up to a 10-7 dilution

37
to reduce the microbial load. 0.5ml of the suspension from resulting 10-4 and 10-6 dilutions were

plated onto nutrient agar and PDA media using the pour plate method. In this method, 0.5ml of the

dilution was added to a sterile, labeled petri dish, and nutrient agar and PDA were introduced. The

mixture was swirled both clockwise and counterclockwise to ensure homogeneity. The plates were

then left to solidify and incubated at 37°C for 2-3 days.

In this study, the serial dilution and pour plate techniques were employed to isolate bacterial

colonies from soil samples. Colonies were counted using dilutions of 10-4 and 10-6 on two different

agar media, namely Nutrient agar and Potato dextrose agar. The aim of the experiment was to

determine the bacterial colony count and observe their morphological properties.

3.3.1. PURIFICATION (SUBCULTURING) OF BACTERIAL ISOLATES

The streak plate method is commonly used to create pure cultures while subculturing bacterial

isolates. This technique involves streaking a loopful of the bacterial culture across the surface of a

Nutrient agar plate in a way that produces a gradient of decreasing bacterial density. Isolated

colonies were obtained from the region with the lowest bacterial density by streaking the loop

across the nutrient agar surface in a specific pattern (four gradient patterns). The colonies from the

plates were continuously subculture four times to produce a pure culture. Aseptic techniques are

employed throughout the streaking process to prevent contamination and ensure the purity of the

resulting cultures.

38
3.4 GRAM’S STAINING TECHNIQUE

The materials used for this test include Slides, Paper tape, Inoculating loop and Gram’s reagents.

Gram staining is a microbiological technique performed to classify bacteria into two groups:

gram-positive (which appear purple or blue) and gram-negative (which appear pink or red). This

process was done by preparing a smear of a pure culture on a grease-free slide by mixing a small

amount of the bacterial growth with a drop of distilled water. The smear was then allowed to air

dry and was heat-fixed by passing the slide through a flame multiple time. The heat-fixed smear

was covered with crystal violet, the primary stain, for one minute and then gently rinsed with tap

water. Next, Gram’s iodine was added as a mordant and allowed to react for one minute before

being washed off. Acetone alcohol, the decolorizing agent, was applied and immediately washed

off. The smear was then counterstained with safranin for one minute and washed off again. After

air drying, a drop of oil immersion was placed on the stained smear, which was then viewed under

a high-power (100X) objective lens of the microscope. A pink or reddish color indicated gram-

negative bacteria, while a purple color indicated gram-positive bacteria following the standard

method conducted in (Aneja, 2003; Lavakush et al., 2012; Majeed et al., 2015).

3.5 BIOCHEMICAL CHARACTERIZATION OF THE ISOLATES

3.5.1 CATALASE TEST

The catalase test detects the enzyme catalase, which breaks down hydrogen peroxide (3%) into

oxygen and water. Following the method of (Schaad, 1992; Aneja, 2003; Lavakush et al., 2012).

To perform the test, a drop of hydrogen peroxide (3%) is placed on a clean glass slide. A 24-hour

old bacterial colony is picked with a sterile loop and mixed into the hydrogen peroxide drop. The

39
mixture is then observed for bubble formation. The appearance of bubbles within a few seconds

indicates a positive result, while no bubbles indicate a negative result.

3.5.2 COAGULASE TEST

The coagulase test is used to differentiate Staphylococcus aureus (coagulase-positive) from

Staphylococcus epidermidis and Staphylococcus saprophyticus (coagulase-negative), collectively

known as coagulase-negative staphylococci (CONS). This enzyme causes plasma to clot by

converting fibrinogen to fibrin. The slide test method involves emulsifying a colony of the test

organism in a drop of distilled water on each end of a slide to create two thick suspensions. A

loopful of human plasma is added to one suspension, which is then gently rocked while no plasma

is added to the second suspension to differentiate any granular appearance of the organism from

true coagulase clumping. Clumping indicates a positive result, while the absence of clumping or

change indicates a negative result.

3.5.3 INDOLE TEST

The indole test identifies organisms that produce the enzyme tryptophanase, which hydrolyzes the

amino acid tryptophan, releasing indole which then accumulates in the medium. A sterile wire

loop is used to inoculate the test organisms into test tubes containing 10 ml of Tryptone

bacteriological broth, which are then incubated for 24 hours at 37°C. After incubation, 0.5 ml of

Kovac’s reagent is added to the tube and allowed to stand for 15 minutes. A red ring at the surface

indicates a positive result, while the absence of a red ring indicates a negative result.

40
3.5.4 OXIDASE TEST

The oxidase test identifies bacteria that produce the enzyme cytochrome c oxidase, which is part

of the electron transport chain in many aerobic organisms. To performed the test: A piece of filter

paper was cut into four equal halves. One of these halves was placed on a petri dish and saturated

with oxidase reagent tetramethyl-p-phenylenediamine (TMPD). Using an inoculating loop, the

organism was then applied to the saturated paper and observed for any color change. If the bacteria

produce cytochrome c oxidase, the enzyme will oxidize TMPD, resulting in a blue-purple color

within a few seconds to a minute. A blue-purple color indicates a positive result, while no color

change indicates a negative result.

3.5.5 SUGAR FERMENTATION TEST

This test assesses an organism’s capability to ferment glucose and convert the end product of

glycolysis, pyruvic acid, into gaseous byproducts. It is frequently employed to identify Gram-

negative enteric bacteria, which ferment glucose universally but vary in their ability to produce

gas. Following the method described by (Aneja, 2003; Lavakush et al., 2012) to conduct the test:

1g of the sugar (glucose, lactose, sucrose, fructose, mannitol, and galactose) was added to 100ml

of peptone water. 2-5 drops of phenol red stain were added and dissolved the sugar thoroughly.

The solution was transferred into test tubes after adjusting with distilled water. An inverted

Durham’s tube was placed into each test tube and ensured a secure seal. The tubes were sterilized

for 15 minutes, then cool to around 40°C. After sterilization, the cultured organisms were

inoculated into each tube and incubated at 28°C-35°C for 24-48 hours. A color change of the

medium from purple to yellow after 24 hours indicated acid production due to sugar fermentation

by the organisms, while retention of the red color indicated a negative reaction. Gas production

41
was observed by the presence of bubbles on the surface of the medium and the upward movement

of the inverted Durham’s tubes.

3.5.6 STARCH HYDROLYSIS TEST

This test detects the presence of the enzyme amylase, which breaks down starch into smaller

glucose units. As described by (Aneja, 2003; Lavakush et al., 2012), Starch agar medium was

prepared according to the manufacturer’s instructions and poured into petri dishes, where it was

allowed to solidify. Once solidified, Pure cultures of test organisms were then inoculated onto the

plates by streaking with a sterile loop. The plates were subsequently incubated, typically at 37°C,

for 24-48 hours. After incubation, Iodine solution was flooded onto the plates, reacting with

unhydrolyzed starch to produce a dark brown color. The appearance of a clear zone around

bacterial growth indicated starch hydrolysis and a positive reaction, while the absence of a clear

zone indicated no hydrolysis and a negative test result.

3.5.7 CITRATE TEST

This test evaluates an organism’s capability to utilize citrate as its sole carbon source. Simmon’s

citrate agar slants were prepared in bijou bottles according to manufacturer instructions. Using a

sterile straight wire and loop, the surface of the Simmon’s citrate agar slope was streaked with the

test organisms, and the butt was inoculated by stabbing. The tubes were then incubated at 35°C

for 48 hours. A bright blue color in the medium indicates a positive test, while no color change

indicates a negative result.

42
3.5.8 SULPHUR REDUCING TEST

This test assesses an organism’s ability to reduce sulfur using the enzyme thiosulfate reductase,

producing hydrogen sulfide gas. It is utilized to differentiate Gram-negative enteric bacilli based

on their ability to produce sulfide. KIA (Kligler Iron Agar) was prepared following the

manufacturer description adding agar for solidification. 10ml of the agar was dispensed into test

tubes and labelled correctly. The test tubes were sterilized and allow to cool upright on a rack

without slanting. Inoculation of the pure culture organism was done by stabbing the inoculating

loop halfway through the medium with the organism. The medium was incubated for 2-7 days.

After incubation, a color change from brown or chocolate to black indicate positive test, while no

color change shows negative test.

3.5.9 MR-VP TEST (METHYL RED VOUGES PROS KAVER TEST)

This test detects the production of acetoin, a neutral end product of glucose fermentation, by

specific organisms. This test detects the production of acetoin, a neutral end product of glucose

fermentation, by specific organisms. Following the method described by (Aneja, 2003; Lavakush

et al., 2012), various test organisms were inoculated into multiple test tubes containing 5 ml of

prepared MR-VP broth medium and then incubated for 24-48 hours at 37°C. After incubation,

Methyl red indicator and α- naphthanol were added to the test tubes and observed for a color

change. A red color indicates a positive result, while a yellow color indicates a negative test result.

43
3.6 SCREENING BACTERIAL ISOLATES FOR PLANT GROWTH-PROMOTING

ACTIVITY

3.6.1 AMMONIA PRODUCTION TEST

This test determines the ability of tested organisms to produce ammonia by degrading amino acids.

Peptone broth medium (4%) was prepared as per manufacturer instructions. After sterilization and

cooling, 50µl of various bacterial cell suspensions were inoculated into 15ml of peptone broth in

screw-capped bottles. The inoculated broth was then incubated at 25-37°C for 72 hours. After

incubation, 1ml of Nessler’s reagent was added to each broth. The development of a yellow to

brown precipitate indicated the presence of ammonia and confirmed a positive result (Cappucino

and Sherman, 1992; Geetha et al., 2014).

3.6.2 AUXIN (IAA) PRODUCTION ASSAY

Indole acetic acid (IAA) production is a key characteristic of rhizosphere bacteria that enhances

and promotes plant growth. This test was used to screen rhizobacterial isolates for their ability to

produce auxin (IAA), following the methods described by (Borrow et al., 1955; Sawar and Kremer,

2008; Sherpa et al., 2021). A sterile peptone yeast extract broth (containing 10g peptone, 3g beef

extract, 5g NaCl, 0.204g L-tryptophan, 1L distilled water, and adjusted to pH 7) was prepared. 5ml

of the broth was dispensed into 15ml culture tubes, and a 50µL aliquot of bacterial cell suspension

was added to each tube. The tubes were incubated at 28°C for 72 hours. After incubation, 1.5ml

of the broth culture was transferred to 2ml Eppendorf tubes and centrifuged at 13,000 rpm for 10

minutes and the supernatant was collected. To 1ml of the supernatant, 1ml of Salkowski reagent

(prepared by mixing 150ml H2SO4, 250ml distilled water, and 7.5ml of 0.5M FeCl3 solution) was

added. The mixture was then incubated at 37°C for 1 hour. A red color in the medium indicated

44
IAA production by the isolates, while no color change indicated a negative result. The indole

quantity was measured using a spectrophotometer (721A England lab science) at 530nm.

3.6.3 PHOSPHATE SOLUBILIZATION

This test was used to screen rhizobacterial isolates for their ability to solubilize phosphorus. The

methodology and observations for testing phosphorus solubilization using Pikovskaya’s agar

medium are presented here (Pikovskaya, 1948; Geetha et al., 2014). Pikovskaya’s agar medium

was prepared by mixing 10g of glucose, 5g of calcium phosphate, 0.5g of ammonium sulfate, 0.2g

of potassium chloride, 0.1g of magnesium sulfate, 0.5g of yeast extract, 15g of agar, and 1000ml

of distilled water. A 24-hour broth culture of the bacterial isolates was spot-inoculated onto the

surface of the agar by gently placing the loop on the surface and then streaking out. The plates

were incubated at 28°C for 96 hours. After incubation, the plates were examined for the presence

of a clear zone around the bacterial colony, indicating phosphorus solubilization. The

solubilization zone was measured by determining the diameter of each halo and its respective

colony using a ruler. The average diameter for each plate was calculated and used to determine the

phosphate solubilization index (PSI) for each strain using the formula:

PSI = Dh/Dc

where (Dh) is the average diameter of halos around the colonies and (Dc) is the average diameter

of the colonies. Phosphate solubilization was categorized as low (if PSI < 2), medium (if 2 ≤ PSI

< 3), or high (if PSI ≥ 3) as described by (Batista et al., 2021).

3.6.4 PROTEASE AND α- AMYLASE PRODUCTION

45
Enzymes play a crucial role in various biological processes; α-amylase and protease are two

important enzymes with diverse functionalities. This test was used to screen rhizobacteria isolates

for the ability to produce protease and α-amylase enzymes. During the process, bacterial cell

suspension of the isolates was prepared and one loopful of the suspension was streaked on two

different agar plates. The first plate used was a starch agar plate, containing peptone 5g, beef extract

3g, soluble starch 10g, agar 15g and distilled water 1000mL. The second plate was a skim milk agar

plate, consisting of skim milk 100g, peptone 5g, agar 15g and water 1000mL. These media were

selected as they provide suitable substrates for detecting the enzymes activity, respectively. After

streaking the bacterial suspension onto the respective plates, the plates were incubated at 28°C for

48hours. After incubation, the plates were carefully examined for the presence of a clear zone

around the streaked area. A distinct clear zone surrounding the streaked area indicates the hydrolysis

of starch by α-amylase, confirming its production. In contrast, the absence of a clear zone indicates

the lack of α-amylase production by the bacterial cells. On the skim milk agar plates, the presence

or absence of a clear zone indicates the production of protease enzyme by the bacterial cells. A

distinct clear zone surrounding the streaked area suggested the hydrolysis of milk proteins by

protease, indicating its production. The absence of a clear zone indicates the absence of protease

production.

3.7 MOLECULAR IDENTIFICATION OF THE SELECTED PSB AND

METAGENOMICS

46
3.7.1 BACTERIAL DNA EXTRACTION

Bacterial samples showing promising results in the screening test were subjected to molecular

identification for DNA extraction. The bacterial genomic DNA was extracted with the help of

ZymoBIOMICS™ DNA Miniprep Kit. The procedure for the DNA extraction is as follows: A

volume of 400 µl of Genomic Lysis Buffer is added to 100 µl of the isolate. The mixture is

thoroughly mixed by vortexing for 4-6 seconds and allowed to stand for 5-10 minutes at room

temperature. The mixture is then transferred to a Zymo-Spin™ IICR Column placed in a

Collection Tube and centrifuged at 10,000 x g for one minute. The Collection Tube and its flow-

through are discarded. The Zymo-Spin™ IICR Column is moved to a new Collection Tube, and

200 µl of DNA Pre-Wash Buffer is added, followed by centrifugation at 10,000 x g for one minute.

Next, 500 µl of g-DNA Wash Buffer is added to the column and centrifuged again at 10,000 x g

for one minute. The spin column is then transferred to a clean microcentrifuge tube, and at least

50 µl of DNA Elution Buffer or water is added. The mixture is incubated for 2-5 minutes at room

temperature and centrifuged at 10,000 x g for 30 seconds to elute the DNA. The eluted DNA can

be used immediately for molecular applications or stored at -20ºC for future use. The quantity of

DNA yield from the extract was measured using a Nanodrop Spectrophotomer. The yield was later

subjected to Polymerase Chain Reaction for amplification using the universal bacterial 27F

forward primer and 1492R reverse primer.

3.7.2 POLYMERASE CHAIN REACTION

47
The bacterial 16S DNA genes were amplified by polymerase chain reaction (PCR) using two

bacterial universal primers: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-

CGGTTACCTTGTTACGACTT-3′). PCR was conducted in a total volume of 50 µL in a PCR

tube, containing 25µL of the 2XMM ZymoTaq PreMix, 5µL of the diluted 27F bacterial universal

forward primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′), 5µL of 1492R reverse primer (5′-

CGGTTACCTTGTTACGACTT-3′), 5µL of Nuclease free water (Injection water) and 10µL of

the DNA extract. The reaction components are gently mixed by pipetting up and down, and the

tubes are briefly centrifuged to ensure all components are at the bottom. PCR Reaction were carried

out using the Mastercycler gradient (Eppendorf, India) with the following conditions: initial

denaturation at 95°C for 5 minutes, followed by 35 cycles of 95°C for 30 seconds, 49°C for 30

seconds, and 72°C for 30 seconds, with a final extension at 72°C for 10 minutes. The amplicon

obtained was subjected to Electrophoresis. The amplified products were stored at 4°C for

immediate use or at -20°C for long-term storage (Ehmann et al., 1977; Batista et al., 2021)

3.7.3 ELECTROPHORESIS

A 1% agarose gel solution is prepared by dissolving agarose in 1X TAE buffer and heating the

mixture until fully dissolved. The solution is cooled to approximately 60°C and poured into a gel

casting tray with a comb to create wells, then allowed to solidify. DNA samples are mixed with a

6X loading dye and 11X SYBR green to achieve a final concentration of 1X, which helps visualize

the samples during loading and adds density to ensure they sink into the wells. The SYBR Green

intercalates with the DNA for visualization. The solidified agarose gel is placed into an

electrophoresis tank filled with 1X TAE buffer until submerged. The prepared DNA samples are

carefully loaded into the wells of the agarose gel using a pipette, and a DNA ladder or molecular

weight marker is included in one of the wells as a reference for fragment size. The electrophoresis

48
tank is connected to a power supply, and the gel is run at 80-120V. The duration of electrophoresis

depends on the gel size and desired separation resolution, typically ranging from 30 minutes to 1

hour. Negatively charged DNA fragments migrate towards the positive electrode (anode), with

smaller fragments moving faster than larger ones. After electrophoresis, the gel is stained with an

appropriate DNA stain such as ethidium bromide or a safer alternative like SYBR Safe for about

15-30 minutes, depending on the stain used. The stained DNA fragments are visualized using a

UV transilluminator or a blue light transilluminator, and an image is captured for documentation

(Gallagher and Wiley, 2008). The bands obtained are viewed using a LI-COR D-digit Imager.

3.7.4 ILLUMINA SEQUENCING METHOD

The sequencing libraries were prepared using the Illumina DNA Prep Kit, employing both PCR

and tagmentation methods. IDT 10bp UDIs were included in the library preparation to ensure

accurate sample identification. The libraries were designed with a target insert size of 320bp, and

no additional DNA fragmentation or size selection steps were incorporated. An Illumina NovaSeq

6000 sequencer was used for the Illumina sequencing. For each sample, 2×151 bp paired-end reads

were generated using this platform. To enhance output and efficiency, multiplexed shared-flow-

cell runs were performed. The sequencer was operated according to established procedures and

guidelines. Bcl-convert1 (version 4.1.5) was employed for post-sequencing tasks, including

demultiplexing, quality control, and adapter trimming. Demultiplexing facilitated the separation

of sequencing reads based on their unique dual indices (UDI), ensuring accurate assignment to the

correct samples. Quality control measures were implemented to ensure the reliability and integrity

of the sequence data. Additionally, adapter trimming was conducted to remove any residual

adapter sequences that could impact subsequent analyses.

49
3.7.5 COMPREHENSIVE AND FUNCTIONAL GENOME ANALYSIS OF Acinetobacter

baumannii strain OYA S31

Reads for the Bacteria Acinetobacter baumannii strain OYA S31 with the accession number

NZ_JAWPGZ010000000 were submitted to the comprehensive genome analysis service at

PATRIC. For genome annotation, two tools were utilized: the NCBI-Prokaryotic Genome

Annotation Pipeline (PGAP) and the RAST toolkit (RAST tk) (Brettin et al., 2015). Based on the

annotation statistics and a comparison to other genomes in PATRIC within this same species, this

genome appears to be of good quality. Details of the analysis, including genes of interest (Specialty

Genes), a functional categorization (Subsystems), and a phylogenetic tree (Phylogenetic Analysis)

are provided below. Gene prediction involved mapping genes to pathways and comparing them

with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.

3.8 FIELD TRIAL WITHIN CONTROLLED GREENHOUSE ENVIRONMENT

3.8.1 MAIZE SEED BACTERIZATION

The seed used for conducting the field trial of the PGB is Maize (Zea mays). Maize seeds were

subjected to surface sterilization by immersing them in a 15 mL solution of 3% hypochlorite for 5

minutes. Following this, the seeds were thoroughly rinsed 10 times with phosphate buffer. A broth

solution containing the IS-13 isolate was prepared and centrifuged at 3000 rpm for 15 minutes to

separate the bacterial cells from the supernatant. The cell pellets were collected, the supernatant

was discarded, and the pellets were rinsed with 1 mL of buffer and mixed well. The mixed bacterial

cell pellets were then placed into sterile test tubes. The bacterial suspension concentration was

assessed using Mac-farland standards of varying concentrations (0.5, 1.0, 2.0, 3.0, and 4.0). The

maize seeds were bio-primed by mixing them with the bacterial cell pellet mixture and adding 1 g

50
of autoclaved kaolin. The bio-primed seeds were placed in petri dishes and incubated for 12 hours.

For the control group, seeds that were not bio-primed but only surface sterilized and rinsed with

sterile phosphate buffer were used. After the 12-hour incubation, both bio-primed and control

seeds were planted in pots containing 2 kg of autoclaved soil as described by (Aziz et al., 2015;

Roslan et al., 2020).

3.8.2 PLANT EXPERIMENT

The soil for the experiment was sieved through a 2-10mm mesh to remove debris and then

sterilized by autoclaving to eliminate contaminants. Sterile plastic container were filled with 10kg

of autoclaved soil, providing a depth of about 45cm. Maize seeds, previously treated with bacterial

cultures, were sown in these pots. The experiment was conducted for the IS-13 (Pseudomonas

fluorescens) and OYA S31 (Acinetobacter baumannii) with 14 seeds planted in each pot. A control

group, consisting of sterile seeds, was established for comparison to assess the effects of bacterial

inoculation. The plants were placed in a greenhouse and maintained under varying conditions of

temperature, relative humidity, and photoperiod for 14 days. After 14 days, plants from pots were

collected. Various growth indicators were measured, including percentage germination, root

length, root branching and diameter, plant height, plant girth, leaf length, leaf width, leaf color.

Data on these parameters were collected over a period of 7 weeks as described by (Aziz et al.,

2015).

3.9 STATISTICAL ANALYSIS OF MEASUREMENT

SPSS version 27 software, provided by IBM Corp. in Armonk, New York, USA, was used for all

data analysis. The analysis employed Mean ± SE, t-test, and One-way ANOVA (Analysis of

51
Variance) to examine significant variations between variable components. To compare differences

between treatments, the mean and ANOVA test with a P value of 0.05 were used.

52
 CHAPTER FOUR

5.0. RESULT

5.1.COLONY COUNT AND MORPHOLOGICAL CHARACTERISTICS OF

BACTERIAL ISOLATES

This chapter presents and discusses the study's findings. The total number of naturally occurring

bacteria in different conditions are highlighted in Table 4.1. The data on the total viable bacteria

count shows variations in colony counts across different agar media and dilutions. Table 4.2. offers

a comprehensive description of the morphological characteristics of the bacterial isolates used in

this study, emphasizing their diversity. The morphological traits of the bacterial isolates vary

according to several parameters. The isolates range in size from small to large, each exhibiting

distinct colony shapes and surface features such as textures and patterns. These differences

highlight the diversity of the isolated bacteria and may suggest variations in their physiological

and biochemical properties.

4.2 GRAM STAINING AND BIOCHEMICAL CHARACTERISTICS OF BACTERIAL

ISOLATES

The Table 4.3 below provides information on the biochemical properties and gram staining of the

bacterial isolates under study. These characteristics are essential for understanding the

physiological properties of the isolates and assessing their potential effects on phosphorus

mobilization and solubilization in the soil, as well as their impact on the growth rate of maize

plants. The table presents the results of various biochemical tests, including details on gram

reaction, catalase, and coagulase activities. These characteristics provide insights into the

53
metabolic capabilities of the bacterial isolate and its potential interactions with soil and maize

plants.

Plate 4.1. displays the results of the microscopic analysis of bacterial isolates after gram staining

at X100 magnification. This visualization provides detailed insights into the cellular properties of

the isolates, enhancing the characterization of the bacterial strains.

4.3 SOIL AGRONOMIC PARAMETERS (RESULTS FOR SOIL ANALYSIS)

The Table 4.5 below presents data on various soil factors, shedding light on soil properties and

their potential impact on interactions with bacterial isolates and maize plants. These factors reveal

the soil's physical, chemical, and agronomic characteristics, including its fertility, nutrient

availability, and overall suitability for maize plant growth. However, the Soil sample E was used

in conducting this study.

54
Table 4.1: Total Viable Bacteria Count

AGAR MEDIA DILUTIONS COLONY COUNT

(CFU/g)

Nutrient Agar 10-4 1.41 × 106 cfu/g

Nutrient Agar 10-6 7.5 × 108 cfu/g

Potato dextrose Agar 10-4 4.8 × 105 cfu/g

Potato dextrose Agar 10-6 4.1 × 108 cfu/g

KEY: CFU/g = Colony forming unit per 1gram of soil

55
 Table 4.2: Morphological Characteristics of Bacterial Isolates on Nutrient Agar Plates
COLONY SHAPE

ELEVATION
ISOLATES

TEXTURE

SURFACE
COLOUR
SIZE
a

IS-13 Medium Circular Light Flat Moist Smooth

Cream
IS-10a Medium Irregular Cream Raised Moist Smooth

IS-14b Medium Irregular White Raised Moist Smooth

IS-4a Small Circular Cream Raised Moist Smooth

IS-9 Small Circular Cream Raised Moist Smooth

Note. Several organisms were isolated but Isolates (IS-13, 10a,14b, 4a, and 9) represent the

bacterial isolates that was selected and further used for the study.

56
 Table 4.3: Gram Staining and Biochemical Characteristics of Bacterial Isolates

HYDROLYSISI

AGAR
HYDROLYSIS
COAGULASE

(SUSPECTED
ORGANISM)
INFERENCE
GRAM RXN

CATALASE
ISOLATES

SULPHUR

OXIDASE
CITRATE
STARCH

UREASE
INDOLE

INDOLE
MR

VP

IS-13 -ve + + + − − − − BLOOD

− + − + + Pseudomonas

Short fluorescens
rods

IS-10a +ve + + − − − + − − − − + + Brucella spp.

Rods

IS- +ve + − − − + + − + − − + + Bacillus cereus

14b
Rods

IS-4a +ve + + − − + + − − − − + + Bacillus subtilis

Rods

IS-9 +ve + − − − + − − + + − + + Alcaligenes

faecalis
Rods

Suspected organisms: IS-13 is Pseudomonas fluorescens, IS-10a is Brucella spp., IS-14b is Bacillus
cereus, IS-4a is Bacillus subtilis and IS-9 is Alcaligenes faecalis.

57
Table 4.4: Sugar Fermentation Result and Gas Production

ISOLATE LACTOSE GLUCOSE SUCROSE MANNITOL GAS

IS-13 − + − − −

IS-10a − + − − −

IS-14b + + + + −

IS-4a − + + + −

IS-9 − + + − −

58
Plate 4.1: Microscopic examination of bacterial isolates after gram staining, viewed under a

magnification of X100.

59
4.4 SCREENING FOR PLANT GROWTH PROMOTING ACTIVITIES

4.4.1 GROWTH PROMOTING TRAIT: AUXIN PRODUCTION ASSAY OF PGPR

Table 4.6 presents the results of PGPR auxin (indole-3-acetic acid, IAA) production. This data

provides insight into how these microorganisms might affect the growth rate of maize plants. The

study reveals differences in auxin production levels among the various isolates, which could be

linked to their ability to impact maize plant growth rates. The control value using Salkowski

reagent serves as a reference, indicating the presence and production of auxin by the PGPR.

4.4.2 NUTRIENT SOLUBILIZATION TRAIT: PHOSPHATE SOLUBILIZATION

ASSAY OF PGPR

Table 4.7. presents the results of the phosphate solubilization assay. This analysis helps assess the

potential impact of these bacteria on the availability of phosphorus in the soil. The results are

expressed as the Phosphate Solubilization Index (PSI), which indicates the extent of phosphate

solubilization. This trait significantly enhances the maize plant's access to phosphorus, potentially

influencing its growth rate. PSI values vary according to each isolate's ability to solubilize

phosphate.

4.4.3 PECTINASE PRODUCTION

The result of the experiments on pectinase and α-amylase production is display in Table 4.8 These

results indicate whether the PGPR isolates produce α-amylase and pectinase. These enzymes play

a role in nutrient release and the breakdown of starch and pectin, respectively, which can influence

the soil-plant interactions. The patterns of enzyme production vary among the different isolates.

60
Table 4.5: Soil Parameters Result avail p (mgkg)

Nitrogen g/kg

Mg (cMol/kg)
Sample name

Ca (cMol/kg)
Na(cMol/kg)
K(cMol/kg)
EC (dS/m)

OM (g/kg)
OC (g/kg)
pH (H20)

A 7.95 0.342 32.4 39.01 67.32 3.22 2.48 0.64 7.02 3.43

B 7.71 0.442 119.42 29.7 51.08 2.84 1.96 0.54 6.86 2.42

C 6.13 0.274 18.55 13.6 23.39 1.68 1.12 0.43 2.46 1.88

D 7.16 0.176 49.42 12.88 22.15 1.48 0.98 0.36 3.46 2.14

E 6.16 0.401 24.71 10.38 17.38 1.12 1.86 0.38 2.86 1.88

F 6.48 0.194 12.32 3.58 6.18 0.96 1.02 0.29 2.12 1.62

G 5.38 0.219 26.74 6.08 10.45 1.04 1.88 0.30 2.34 1.84

H 7.84 0.256 214.13 31.41 54.15 3.40 2.42 0.42 6.12 2.98

I 6.82 0.774 63.84 55.68 96.00 2.98 2.06 0.59 4.28 1.82

KEY: Ph- potential of hydrogen, EC- Electrical conductivity, OC- Total Organic Carbon, OM-

Organis Matter. cMol means centimoles of positive charge per kilogram of soil.

61
Table 4.6: Auxin Production Result

ISOLATES AUXIN (IAA) AMMONIA

IS-13 + −

IS-10a + +

IS-14b + −

IS-4a + −

IS-9 + −

62
Table 4.7: Phosphate Solubilization

ISOLATES RESULTS P-SOLUBILIZATION INFERENCE

INDEX (PSI)

IS-13 + 8 High

IS-10a + 4 High

IS-14b + 3.75 High

IS-4a + 3.6 High

IS-9 + 3.33 High

OYA S31 + 4.22 High

NB: LOW= (PSI < 2), MEDIUM= (2 ≤ PSI < 3), HIGH= (PSI ≥ 3).

KEY: Present (+), Absent (−)

63
Plate 4.2: Phosphate solubilization. The presence of a zone of clearance indicates that the bacterial

isolates have successfully solubilized phosphate.

64
 P- Intensity
8
7
6
5
4
3
2
1
0
IS-13 IS-4a IS- IS- IS-9 OYA
14b 10a S31
PSI Index 8 3.6 3.75 4 3.33 4.2
Isolates

PSI Index

Figure 4.1: A stacked bar chart depicting the solubilization of phosphate intensity produced by

each PGPR isolate, the 1S-13 has the highest among other PGPR screened.

65
Table 4.8: Pectinase Production Result.

ISOLATES PECTINASE
(mm)

IS-13 30mm

IS-10a 15mm

IS-14b 15mm

IS-4a R

IS-9 17mm

66
Plate 4.3: Production of protease enzyme by bacteria cells on skimmed milk agar.

67
4.5 DNA EXTRACTION YIELD

The amount of DNA yield obtained was measured using a Nanodrop Spectrophotometer to assess

purity levels based on A260/230 and A260/280 ratios. IS-13 demonstrated the highest DNA yield

of 109.5 µg/µl with purity levels of 1.89 for A260/280nm. Table 4.9 illustrates the intensity and

the purity of the DNA extract was measured at an absorbance of 260nm, 280nm and 230nm. The

ratio value lower than 1.7 for A260/280nm shows that the sample is contaminated with protein

while ratio value below 1.8 for A260/230nm shows contamination with organic compound like

phenol, alcohol or carbohydrates.

4.6 COMPREHENSIVE AND FUNCTIONAL GENOME OF Acinetobacter baumannii

strain OYA S31

4.6.1 GENOME ASSEMBLY FEATURES

The genome of bacterium OYA S31 was assembled automatically, resulting in 33 contigs, an

estimated genome length of 3,882,953 bp, and an average G+C content of 38.78%. The N50 length,

which is defined as the shortest sequence length at 50% of the genome, is 506,077 bp. The L50

count, which is defined as the smallest number of contigs whose length sum produces N50, is 3 as

detailed in Table 4.10.

68
 Table 4.9: DNA Extract yield and purity level

ISOLATES µg/µl A260/A280 A260/A230

(nm) (nm)

IS-13 109.5 1.89 0.69

OYA S31 60 1.90 0.97

IS-10a 84.1 1.89 0.60

IS-14b 49.2 1.93 0.92

The Table above shows that the DNA extract has no contamination with protein.

69
Table 4.10: Assembly Features

ASSEMBLY FEATURES

Contigs 33

GC Content 38.78

Plasmids 0

Contig L50 3

Genome Length 3, 882, 953 bp

Contig N50 506, 077

Chromosomes 0

70
4.6.2 GENOME ANNOTATION FEATURES FOR OYA S31

For genome annotation, we used two tools: the NCBI-Prokaryotic Genome Annotation Pipeline

(PGAP) and the RAST toolkit (RAST tk) and assigned the unique genome identifier 2.37742

(Brettin et al., 2015). Gene prediction involved mapping genes onto pathways and comparing them

with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.

According to genetic code 11, this genome is classified under the superkingdom Bacteria and

includes 3 ribosomal RNA (rRNA) genes, 63 transfer RNA (tRNA) genes, and 3,685 protein-

coding sequences (CDS). The annotation identified 2,817 functional proteins and 868 hypothetical

proteins. The proteins with functional assignments included 946 proteins with Enzyme

Commission (EC) numbers (Schomburg et al., 2004), 818 with Gene Ontology (GO) assignments

(Ashburner et al., 2000), and 742 proteins that were mapped to KEGG pathways (Kanehisa et al.,

2016). PATRIC annotation includes two types of protein families (Davis et al., 2016), and this

genome has 0 proteins that belong to the genus-specific protein families (PLFams), and 3,612

proteins that belong to the cross-genus protein families (PGFams). Additionally, strain OYA S31

exhibited an 81% average nucleotide identity (ANI) and an 80% assembly coverage compared to

the Acinetobacter baumannii ATCC 17978 400667.7 type strain in the database. Table 4.11.

present the annotation features for the Acinetobacter baumannii OYA S31 genome.

71
Table 4.11: Annotated Genome Features

ANNOTATED GENOME FEATURES

CDS 3,685

tRNA 63

rRNA 3

Repeat Regions 52

Partial CDS 0

Miscellaneous RNA 0

72
Table 4.12: Protein Features

PROTEIN FEATURES

Hypothetical proteins 868

Protein with functional assignments 2,817

Proteins with EC number assignments 946

Proteins with GO assignments 818

Proteins with Pathway assignments 742

Proteins with PATRIC genus-specific 0

family (PLfam) assignments

Proteins with PATRIC cross-genus 3,612

family (PGFAM) assignments

73
4.6.3 SUBSYSTEM ANALYSIS

A subsystem comprises a group of proteins that collectively carry out a specific biological process

or form a structural complex. An overview of the subsystems for this genome is illustrated in

Figure 4.2.

4.6.4 PHYLOGENETIC ANALYSIS

The Assemby contigs.fasta files was duplicated and input into the NCBI Blast tool. This powerful

tool was employed to identify significant sequence alignments among the contigs. The closest

reference and representative genomes to were identified by Mash/MinHash (Ondov et al, 2016).

PATRIC global protein families (PGFams) (Davis et al., 2016) were selected from these genomes

to determine the phylogenetic placement of this genome. The protein sequences from these

families were aligned with MUSCLE (Edgaret al., 2004), and the nucleotides for each of those

sequences were mapped to the protein alignment. A total of 11 sequences were chosen based on

their ability to produce meaningful alignments. We employed the neighbor-joining tree method to

construct a phylogenetic tree based on pairwise genetic distances. A maximum sequence difference

of 0.05 was set to ensure that only closely related sequences were included in the analysis. The

phylogenetic tree generated through this analysis revealed the evolutionary relationships among

11 selected sequences. Branch lengths in the tree represent the genetic distances between the

sequences, with shorter branches indicating closer relationships. The joint set of amino acid and

nucleotide alignments were concatenated into a data matrix, and RaxML (Stamatakis, 2014) was

used to analyze this matrix, with fast bootstrapping (Stamatakis et al., 2008) was used to generate

the support values in the tree Fig. 4.3.

74
Figure 4.2: Subsystem pathways.

75
Figure 4.3. Phylogenetic Tree Analysis

76
4.7 STATISTICAL ANALYSIS OF IS-13 ISOLATE AND OYA S31 ON MAIZE GROWTH
(Zea mays)

The agronomical parameters for the Zea mays were measured for 7 weeks after sprouting. This

subsection explores the effects of auxin-producing and phosphate-solubilizing bacteria on various

aspects of maize (Zea mays) development. The study investigated whether modifying soil

microorganisms, specifically those that produce auxin and solubilize phosphate, could improve

plant growth and development. The impact on several plant growth parameters, including plant

height, girth, root length, root branching, leaf length, leaf breadth, and root diameter, was examined.

The findings indicated a significant growth enhancement in maize plants treated with auxin-

producing and phosphate-solubilizing bacteria. This is illustrated in Table 4.15 and Figure 4.5,

which present the growth characteristics for both treatment groups (IS-13 ISOLATE, OYA S31

and CONTROL).

77
Plate 4.4: Image of Plant for the Treatments (A= Control, B= Acinetobacter baumannii OYA S31,

C= IS-13 Isolate). The IS-13 treated maize plant shows a rapid plant growth and sustainability

better than the OYA S31 treatment while the Control shows the lowest. (IS-13 suspected organism

is Pseudomonas fluorescens, OYA S31 is Acinetobacter baumannii).

78
 Table 4.13: Agronomical parameter of the Maize plant (IS-13 and OYA S31 are PGPRs)

Plant % Leaf Leaf Leaf Shoot Root Plant

number length length length Girth
Germination Width
(cm) (cm) (cm) (mm)

WEEK 1 Control 78.6% 4 1.8 26.1 7.4 11 4.53

OYA S31 93% 5 2.1 31.3 8.5 8 5.11
IS-13 100% 5 1.8 37.5 10 11 5.91
WEEK 2 Control 5 2.2 38.9 8.8 11.3 4.35
OYA S31 5 2.2 29.7 7 14.5 6.25
IS-13 5 2.5 40 9.3 19 5.86
WEEK 3 Control 5 2.5 38 8 13.6 5.64
OYA S31 6 2.9 47.3 9.5 12.8 5.98
IS-13 5 3 48 11 12.5 5.63
WEEK 4 Control 5 2.2 39 15 12 4.57
OYA S31 5 2.3 43 10 15.4 4.80
IS-13 5 2 43 13 12 5.35
WEEK 5 Control 6 3 44 13 15 5.20
OYA S31 7 2.5 44 15 15 6.20
1S-13 7 4 49 16 20 6.30
WEEK 6 Control 4 2.5 42 17.3 11 5.27
OYA S31 5 2.8 46.5 17.5 12.5 6.55
IS-13 7 3.3 61.5 18 16 8.79
WEEK 7 Control 5 2.4 48 16 15 6.40
OYA S31 5 3.5 49 21 21 8.34
IS-13 7 3.5 64.5 28.5 25 9.24

79
Table 4.14: Mean and Standard Deviation statistics for the Treatment.

80
4.7.1 PLANT HEIGHT AND GIRTH

Figure 4.5 and Table 4.15 illustrate the effects of the IS-13 isolate on maize plant height and girth.

Plants treated with the IS-13 isolate had an average height of 15.1143 cm, which was greater than

the 12.2143 cm average height of the control plants. Additionally, the treated plants had a larger

girth (6.7257mm) compared to the control plants (5.1371 mm). A two-sample t-test was performed

to assess the statistical differences in shoot height and girth between the treatment groups, as shown

in Table 4.15. The t-test results showed a p-value of 0.430 for plant height, indicating that the

difference was not statistically significant. In contrast, the p-value for plant girth was 0.037

demonstrating statistically significant difference despite being just below the conventional

significance threshold of p < 0.05 which shows the great influence of the PGPR isolate in

strengthen the stem and ensuring a healthier plant growth.

4.7.2 ROOT LENGTH AND BRANCHING

Root length and branching significantly impact a plant's ability to access soil nutrients and water.

Table 4.14. indicates that plants treated with the IS-13 isolate had longer roots with average value

of 16.5000 cm compared to those in the control group with a mean of 12.7000 cm. Additionally,

treated plants exhibited more root length and branching compared to the OYA S31 (14.1714) and

control group. However, the t-test results performed for the treatment of control group and IS-13

group yielded p-values of 0.021 for root, showing a statistically significant differences among

these measurements at significance threshold of p < 0.05 revealing the impact of IS-13 isolate as

a growth promoting tool for ensuring adequate uptake of nutrient and other essential molecules via

the plant root.

81
 MEAN

12

10

0
CONTROL OYA S31 IS-13
Shoot length 7.64 8.32 9.52
Plant girth 5.14 6.18 11.2

Shoot length Plant girth

Figure 4.4: Bar Chart depicting the average value of Plant girth and Shoot length for each

treatment: The IS-13 shows the highest average value followed by the OYA S31 and CONTROL

has the lowest.

82
 MEAN
12
10
8
6
4
2
0
CONTROL OYA S31 IS-13
Root length 5.44 7.38 9.22
Leaf number 6.8 9 11.2
Leaf length 4.22 4.86 6.26
Leaf width 3.48 3.7 4.12

Root length Leaf number Leaf length Leaf width

Figure 4.5: Bar Chart depicting the average value of Root length and Leaf morphology for each

treatment

83
4.7.3 LEAF MORPHOLOGY

Leaf numbers, length and width were measured to assess the impact of the IS-13 isolate on leaf

morphology. Table 4.14 shows that plants treated with the IS-13 isolate had longer leaves with a

mean of 49.0714 cm compared to control plants with a mean of 39.4286 cm and wider leaves

(2.8714 cm) compared to the control (2.3714 cm). However, the t-test results indicated that the

differences in leaf length to be p = 0.248 which shows a non-statistically significant difference in

the treatment while the leaf width indicate a p value of 0.035 showing a statistically significant

differences at p < 0.05. This reveals a large effect of the organism in promoting the leaf

morphology of Maize plant.

84
Table 4.15: Influence of Auxin-Producing and Phosphate Solubilizing Bacteria (IS-13) on Maize

Growth Factors

T-test For Treatment vs Plant Growth Metrics

Growth
Parameters F df p-value

Plant Height 0.668 9.947 0.430

Plant Girth 5.495 8.394 0.037
Root Length 6.997 7.472 0.021
Leaf Number 7.646 10.260 0.017
Leaf Length 1.473 10.387 0.248
Leaf Width 5.645 8.393 0.035

85
 MEAN

30

25

20

15

10

0
Leaf Leaf width Leaf Root Shoot Plant girth
Number length length length
CONTROL 6.8 3.48 4.22 5.44 7.64 5.1371
OYA S31 9 3.7 4.86 7.38 8.32 6.1757
IS-13 11.2 4.12 6.26 9.22 9.52 6.7257

IS-13 OYA S31 CONTROL

Figure 4.6: A stacked bar chart depicting the mean of effect of the treatment on the Plant Metrics.

The blue shows the effect of IS-13 on the plant parameter promoting the plant growth with a large

margin compare to the red showing the effect of OYA S31 on the Maize plant and the green

indicate for CONTROL with a low effect on the plant growth metrics.

86
 CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS

5.1. DISCUSSION

The rhizosphere is the region where interactions between plants, soil, and microorganisms take

place at the plant-root interface (Ding et al., 2019). Plant-growth-promoting rhizobacteria are free-

living organisms that colonize plant roots and positively influence plant growth. These

microorganisms can enhance plant growth and solubilize phosphates through their metabolic

activities. They directly affect plant metabolism by producing hormones and fixing nitrogen (Dos

Santos et al., 2020). This study's report is on the enhancement of Maize growth through seed

bacterization with Plant Growth-Promoting Rhizobacteria (PGPR) and in comparison, with the

organism Acinetobacter baumannii OYA S31 action on the maize similar to the study conducted

by (Akinola et al., 2022; Chabbi et al., 2024). The IS-13 isolate suspected to be Pseudomonas

fluorescens shows a high level of phosphate solubilizing index of 8 PSI and auxin production on

the Maize plant. The presence of the phophate solubilizing gene in the IS-13 isolate promotes the

growth of the Maize plant and auxin level by making phosphate readily available for the plant and

generating essential macronutrient and micronutrient needed for the plant growth and development.

The IS-13 isolate was suspected to be Pseudomonas fluorescens strain from the inference of the

screening in biochemical test and plant growth promoting activity. A recent study by (Khan et al.,

2009; Adedeji et al., 2020; Riaz et al., 2021) reveals the Pseudomonas strains identified as

phosphorus solubilizers due to the production of organic acids and phosphatases, thereby

facilitating the solubilization of phosphorus and other nutrients. The organism also shows a great

potential in converting the ability to convert insoluble inorganic phosphorus compounds to

87
bioavailable forms and stress regulator in plant as similarly observed in a study conducted in

(Paliwoda et al., 2024). Phosphate solubilizing rhizobacteria (PSRB), residing in the plant

rhizosphere, are recognized as effective bio-inoculants due to their capability to enhance the

availability of phosphates like HPO4−2 or dihydrogen phosphate (H2PO4−1) in the soil. These

microorganisms are particularly beneficial for plants under stress conditions similar to the study

conducted in (Notununu et al., 2022). These microorganisms are particularly beneficial for plants

under stress conditions as discussed by (Billah et al., 2019). The Acinetobacter baumannii OYA

S31 also shows phosphate solubilizing effect and was known to be PGPR active but the action is

not as compared to the efficiency of the IS-13 isolate. Plant-based bioassays conducted under

greenhouse conditions demonstrated significant growth improvements in plants inoculated with

IS-13 isolate which shows better performance over the known PGPR OYA S31 in comparison to

the control plant similarly to the performance of Pseudomonas spp. bio-primed maize plant

conducted in (Jarak et al., 2012). The IS-13 (Pseudomonas fluorescens) isolates exhibited rapid

germination in maize plants during the first week with a leaf number of 5 while the Control shows

4 leaf number at week one. The average mean recorded on leaf number of the IS-13 (Pseudomonas

fluorescens) is 5.86 ± 0.404 which is higher than that of OYA S31 (Acinetobacter baumannii) with

average of 5.43 ± 0.297 and the Control group is 4.86 ± 0.261. The mean values obtained from

shoot length are 15.1143 ± 2.53 for IS-13, 12.6429 ± 1.98 for OYA S31 and 12.2143 ± 1.55 for

CONTROL. The mean values obtained for root length over the seven weeks of measurement are

16.5 ± 1.94 for IS-13, 14.1714 ± 1.48 for OYA S31 and 12.7 ± 0.68 for Control. The positive effect

of the OYA S31 organism is somewhat similar to the IS-13 during the earlier stage of the plant but

the IS-13 exhibit a better performance in sustaining the growth and development of the maize plant.

The two organisms prove to be an adequate tool in promoting plant growth and development. The

88
comparison in this study is similar to that conducted in (Akinola et al., 2022), the OYA S31 isolate

demonstrated a competitive performance with IS-13, the PGPR isolate proved to be more effective

in maintaining plant health and stability, providing strength, and inducing disease resistance

compared to Acinetobacter baumannii OYA S31.

5.2. CONCLUSION

As the global population grows, the demand for food continues to rise, elucidating the need for

enhanced and sustainable agricultural productivity. This research shows that seed bacterization is

an environmentally sustainable method for enhancing crop growth, potentially reducing the need

for excessive fertilizer use. Maize as a major cereal crop, widely produced and traded, and is

crucial for food security and industrial applications. It is used in the production of bioethanol, a

renewable energy source that reduces reliance on fossil fuels. The maize rhizosphere is primarily

colonized by various bacteria, fungi, and archaea. Notable bacterial groups include Proteobacteria,

Firmicutes, and Actinobacteria, while common fungal colonizers are Ascomycetes and

Basidiomycetes. These microorganisms are essential for nutrient cycling, promoting plant growth,

and suppressing diseases. Identifying microbial isolates that are native to and well-adapted to

Africa can provide significant benefits for small-scale African farmers. Plant growth-promoting

bacteria used in seed bacterization have demonstrated the potential to improve the growth of Maize,

offering a viable alternative to chemical fertilizers. Specifically, the IS-13 (Pseudomonas

fluorescens) isolate conducted in this study and Acinetobacter baumannii OYA S31 have shown

prominent effect in enhancing the agronomic parameters and crop sustainability of Zea mays.

89
5.3. RECOMMENDATIONS

Plant Growth-Promoting Rhizobacteria (PGPR) provide a promising alternative to chemical

fertilizers, offering a more sustainable agricultural method with reduced environmental impact. It

is crucial to enhance our comprehension of plant-microbe interactions by expanding research on

the specific plant genes that play a role in these processes. Attention should be directed towards

genes involved in signaling pathways, receptor-like kinases, and those associated with symbiotic

relationships with beneficial microbes. Gaining this knowledge will facilitate the development of

crops that are better equipped to establish advantageous associations with microbes.

Advancements in functional genomics techniques, including CRISPR/Cas9 gene editing and RNA

interference, to analyze the functions of specific genes that play a role in plant-microbe interactions

is necessary. Ensure that research and applications resulting from studies on plant-microbe

interactions emphasize sustainability. Develop computational models to forecast how genetic

variations in plants and microbes influence their interactions, aiding in the design of targeted

experiments and enhancing our comprehension of complex interactions. Evaluate the

environmental effects of new crop varieties and strive to create solutions that improve soil health

and minimize reliance on chemical inputs. Adopting sustainable practices will support long-term

agricultural productivity and promote environmental stewardship. By adopting these

recommendations, we can greatly enhance our comprehension of plant-microbe interactions and

use this insight to boost crop resilience, productivity, and sustainability in agriculture.

90
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APPENDICES

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Molecular lab work and analysis performed at Microbiology lab, Adeleke Uni. Osun state and

Prof. OYA Lab, FUOYE, Ekiti State.

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