Comparative Analysis of Microbial Profiles and Trade-offs in Sorghum bicolor

Genotypes (SC1345 and SC265) Under Drought and Salinity Stresses

A Thesis Presented to the Department of Biology

and the Faculty of the Graduate College

University of Nebraska

In Partial Fulfillment
of the Requirements for the Degree
Master of Science

University of Nebraska at Omaha

W
by
IE
Stephanie Cromwell

May 2025
EV

Supervisory Committee:
PR

Paul Ayayee, Ph.D.

Timothy Dickson, Ph.D.
Joe Louis, Ph.D.
 Abstract

Comparative Analysis of Microbial Profiles and Trade-offs in Sorghum bicolor

Genotypes (SC1345 and SC265) Under Drought and Salinity Stresses
Stephanie Cromwell M.S

University of Nebraska, 2025

Advisor: Paul Ayayee, Ph.D.

Climate change is a major global concern, impacting plant productivity and increasing

W
insect pest outbreaks, thereby raising food security challenges in the United States. To

address these issues, NAM nested association mapping lines were used to study sorghum
IE
genotypes’ responses to abiotic stress, focusing on SC1345 and SC265, which were

previously identified as sugarcane aphid-susceptible and -resistant genotypes,

EV
respectively. Although the rhizosphere is critical for plant growth and stress adaptability,

genotype-specific influences on microbial communities under different stresses remain

PR

poorly understood. To investigate these dynamics, 16S rRNA gene sequencing was

conducted to study growth-defense trade-offs and microbial shifts between SC1345 and

SC265 under drought and salinity stress. SC265 invested more in belowground biomass,

while SC1345 prioritized aboveground growth. Significant differences (p < 0.05) were

observed in leaf weight, height, and stem width, while root biomass differences between

genotypes were not significant (p > 0.05). SC1345 exhibited greater structural variability

under different stress conditions, while SC265 displayed stronger integration between

root traits, as indicated by correlation heatmaps. Microbial community structure differed

significantly across drought treatments (p < 0.05), but not under salinity treatments (p >
0.05). SC265 maintained a stable core microbiome that supported root system function,

whereas SC1345 showed a more flexible microbial recruitment strategy. Under high

salinity, SC1345 enriched for Halomonadaceae, while SC265 enriched for

Paenibacteriaceae and Burkholderiaceae. Under drought conditions, SC1345 recruited

Devosiaceae and Caulobacteraceae, while SC265 enriched for Nocardioidaceae and

Propionibacteriaceae. Drought stress produced greater microbial shifts compared to

salinity stress. Bulk soil samples, serving as microbial reservoirs, exhibited significantly

higher microbial diversity and abundance than rhizosphere controls (p < 0.05). Functional

predictions indicated that microbial communities were primarily involved in carbon

W
cycling, while nitrogen-cycling microbes represented a smaller fraction. These findings
IE
highlight genotype-specific strategies in biomass allocation, rhizosphere microbial

assembly, and stress adaptation, providing insights for breeding climate-resilient sorghum
EV
varieties.
PR
 i

Acknowledgments

First and foremost, I give all thanks and glory to God Almighty for His grace, strength,

and guidance throughout my academic journey. Without His divine support, this

achievement would not have been possible.

I am deeply grateful to my advisor, Dr. Paul Ayayee, whose mentorship, encouragement,

and dedication played a pivotal role in my growth as a researcher. His patience, insightful

feedback, and unwavering support have been invaluable throughout my thesis and entire

M.Sc. program. My sincere appreciation also goes to my committee members, Dr.

W
Timothy Dickson and Dr. Joe Louis, for their constructive feedback, support, and

guidance during my research. A heartfelt thank you to Dr. Adenike for her continuous
IE
academic support and the wealth of knowledge she shared with me throughout this
EV
project. Her input made a meaningful difference in the completion of my work.

I gratefully acknowledge the University of Nebraska at Omaha Biology Department for

PR

their departmental funding and consistent support. I also appreciate GRACA (Graduate

Research and Creative Activity grant) for providing grant support, which greatly

contributed to the successful completion of this thesis. Special thanks to the Microbial

Ecology Lab Group (Ayayee Lab) for their collaboration, support, and all the valuable

conversations that enriched this experience. I am also thankful to the Biology Graduate

Student community for their friendship, shared experiences, and constant encouragement.
 ii

To my parents and siblings, thank you for your daily encouragement, prayers, and

unwavering love. I am also truly grateful to my friends and family for their support,

motivation, and well wishes every step of the way.

While I may not have named everyone individually, I deeply appreciate and sincerely

value the support, guidance, and contributions of all those who played a role in this

journey.

W
IE
EV
PR
 iii

TITLE PAGE
ABSTRACT
ACKNOWLEDGEMENTS…………………………………………………...............i
TABLE OF CONTENTS……………………………………………………................iii
LIST OF MULTIMEDIA

a. List of Tables……………………………………………………………………..vi
b. List of Figures……………………………………………………………….........vi
CHAPTER 1: Literature Review
1. Introduction to Sorghum ....................................................................................... 1
1.1 Abiotic Stress Impacts on Sorghum Growth and Development ............................ 2
1.2 Drought Impacts on Sorghum Growth, and Development .................................... 3

W
1.3 Salinity Impacts on Sorghum Growth and Development ...................................... 4
1.4 Effects of Drought and Salinity Pressure on Sorghum Defense and Adaptation ... 6
1.5 Rhizosphere Microbial Influence on Drought and Salinity Stress......................... 7
IE
1.6 Drought and Salinity Stress Influence on Rhizosphere Microbial Ecological
Functions ................................................................................................................ 8
EV
1.7 Trade-offs in Sorghum Rhizosphere Responses under drought and
salinity stress .......................................................................................................... 9
1.8 Potential for Microbiome-Based Solutions.......................................................... 10
1.9 Significance of the current study ......................................................................... 10
PR

CHAPTER 2
Abiotic Stress Impacts on Sorghum Genotypes' Morphology Measurements and
Productivity .......................................................................................................... 13
Abstract ................................................................................................................ 13
2.1 Introduction .......................................................................................................... 14
2.2 Materials and Methods ......................................................................................... 16
2.2.1 Plant Material, Growth Conditions, and Stress Treatments ................................. 16
2.2.2 Measurement of Plant Growth and Biomass Parameters ..................................... 17
2.2.3 Statistical Analysis ............................................................................................... 18
2.3 Result ..................................................................................................................... 18
2.3.1Above and belowground Biomass Response to Stress Treatments ...................... 18
2.4 Discussion ............................................................................................................... 22
 iv

2.5 Conclusion .............................................................................................................. 25

CHAPTER 3
Comparative Study of Rhizosphere Responses to Drought and Salinity Stress in
Sorghum bicolor Genotypes................................................................................... 30
Abstract ................................................................................................................... 30
3.1 Introduction ............................................................................................................. 32
3.2 Materials and Methods ........................................................................................... 35
3.2.1Experimental setup and treatment conditions ....................................................... 35
3.2.2 Collections of samples for DNA sequencing: ...................................................... 36
3.2.3 Data analysis…………………………………………………………………… 37
3.3 Results .................................................................................................................. 39

W
3.3.1 Impact of treatments on rhizosphere diversity and richness ................................ 39
3.3.2 Impact of treatments on rhizosphere community composition and ecological
functional potential ................................................................................................... 39
IE
3.4 Discussion ............................................................................................................... 43
3.5 Conclusion ……………..……………………………………………………….48
EV
CHAPTER 4
Differential Assembly of Rhizosphere and Bulk Soil Microbiomes............................. 54
Abstract ......................................................................................................................... 54
4.1 Introduction .............................................................................................................. 55
PR

4.2 Method ..................................................................................................................... 56

4.2.1 Experimental setup and treatment conditions: ........................................................ 57
4.2.2 Collections of samples for DNA sequencing: ......................................................... 58
4.4.3 Data analysis: .......................................................................................................... 59
4.3 Result ....................................................................................................................... 60
4.4 Discussion ................................................................................................................ 63
4.5 Conclusion ................................................................................................................ 65
CHAPTER 5
5.1 General Conclusion ..................................................................................................... 70
5.2 Biomass Allocation ..................................................................................................... 70
5.3 Rhizosphere Microbial Community Dynamics .......................................................... 70
 v

5.3 Bulk Soil Microbiome Patterns ............................................................................... 71

5.4 Integrating Biomass and Microbiome: Genotype-Specific Trade-Offs .................. 71
5.5 Concluding
Perspective……………………………………………………………………………...72
References…………………………………………………………………………........73

APPENDICES
a. Appendix A …………………………………………………………………………...94
b. Appendix B …………………………………………………………………………...97
c. Appendix C ………………………………………………………………………….106

W
IE
EV
PR
 vi

LIST OF MULTIMEDIA OBJECTS

LIST OF TABLES

Table 3.1 Summary of Mean and Standard Deviation for Shannon, Observed,
and Chao1 indices of SC265 and SC1345 genotypes
Under Drought and Salinity Treatments…………………………………50
LIST OF FIGURES
Figure 2.1 Effects of genotype and treatment on sorghum
growth parameters………............................................................................27
Figure 2.2 Spearman correlations among sorghum growth parameters across
genotypes and treatments………………………………………………....28
Figure 2.3 Aboveground (g) and belowground biomass of two sorghum
genotypes (SC1345 and SC265) under various drought

W
and salinity treatments…………………………………………………....29
Figure 3.1 Microbial diversity (Observed Species, Shannon Index) within samples
IE
varies significantly (P < 0.05) between sorghum genotypes
and treatment levels……………………………………………………....51

Figure 3.2 Microbial community structure and composition across

EV
sorghum genotypes and treatments level……………...............................52

Figure 3.3 Microbial ecological functional predictions using FAPROTAX

between treatment levels based on the relative abundance of
functional potential…………..................................................................53
PR

Figure 4.1 Alpha diversity indices of microbial communities across

sorghum genotypes and soil compartments…………………………….67
Figure 4.2 Microbial community structure and composition across sorghum
genotypes and soil compartments……………………………...……….68

Figure 4.3 Predicted functional profiles of microbial communities at the family

level across sorghum genotypes
and soil compartments……………………….…………………...…….69
 1

CHAPTER 1: Literature Review

Introduction to Sorghum
Sorghum (Sorghum bicolor) is a significant cereal crop in many regions

worldwide due to its adaptability to changing environmental conditions (Elramlawi et al.,

2019). It excels in withstanding and adjusting to various abiotic stresses, including heavy

metals, drought, salinity, and alkaline soils (Tu et al., 2023a). Historically, sorghum

originated from the wild progenitor S. bicolor subsp. verticilliflorum, found in Ethiopia,

Sudan, and West Africa (Ananda et al., 2020). It is the first known C4 plant recognized

W
for its ability to photosynthesize efficiently and utilize nitrogen and water effectively.
IE
Sorghum ranks as the fifth most important multipurpose crop after maize, rice, wheat,

and barley, producing over 64 million metric tons annually (Proietti et al., 2015;
EV
Thilakarathna et al., 2022; Weijde et al., 2013). As a high-carbohydrate and climate-

resilient crop, it plays a crucial role in global food production and security, nourishing

billions of people (Chaturvedi et al., 2023). Sorghum is widely utilized in food products,
PR

animal feed, alcoholic beverages, and biofuels (Stamenković et al., 2020).

It has been used as a raw material for producing cellulosic ethanol, particularly in

Brazil, Australia, and the United States (Hossain et al., 2022a). Compared to other

industrial cereal crops, sorghum requires less water and has a shorter growing season. It

serves as a significant source of bioenergy globally, boasting a fermentation efficiency of

over 90%. These characteristics contribute to sorghum's status as one of the essential

cereal crops cultivated in more than 100 countries, yielding approximately 64 million

tons each year from 45 million hectares of farmland (Guden et al., 2019). Over time,
 2

sorghum grain has gained popularity for human consumption due to its phenolic

compounds, which are utilized for their medicinal properties (Xu et al., 2021). Given the

increasing demand for limited freshwater resources, the expansion of agriculture into

marginal areas, and changing climate patterns (Hemathilake et al., 2022), sorghum has

become an essential crop. These appealing traits make sorghum a promising subject for

various research endeavors aimed at improving food security in a changing climate

(Hossain et al., 2022b), as well as an excellent model for examining evolutionary links

among grass species (Silva et al., 2022).

W
1.1 Abiotic Stress Impacts on Sorghum Growth and Development
Plant development can be disrupted by abiotic stress caused by climate change,
IE
including high temperatures, drought, and salinity. These stressors significantly interfere

with the physiological and biochemical processes that are crucial for plant growth and
EV
reproduction (Zhang et al., 2023a). They reduce plant productivity and affect

reproductive processes, such as pollen viability and seed set, which are particularly
PR

sensitive to changes in temperature and water availability (Gérard et al., 2020). Research

has shown that climate change-related stresses can accelerate phenological events

(Bradley et al., 1999). This acceleration shortens developmental stages and reduces the

time available for carbon assimilation, ultimately impacting crop yields (Minoli et al.,

2019). Moreover, abiotic stress can alter plant interactions with other organisms,

including beneficial microbes and pests, which complicates pest management strategies

and reduces crop resilience (Fadiji et al., 2023). Plants rely on complex stress response

mechanisms, which include activating stress-related genes, accumulating

osmoprotectants, and enhancing antioxidant defenses. The genes activated during

 3

stressful conditions serve a dual purpose: they help protect cells by producing essential

proteins needed for metabolism and regulate other genes that manage the cell's response

to stress (Shinozaki et al. 2022). However, these adaptive mechanisms are often

insufficient under prolonged or extreme stress conditions (Zhu, 2016). Most studies

indicate that climate change will have a significant long-term impact on sorghum yields

compared to short- and medium-term effects. Depending on the region and the specific

climate models used, projections suggest that sorghum yields could decrease by as much

as 41% in the future due to climate change (Khalifa et al., 2023a). Both drought and

salinity are recognized as two major abiotic stresses that significantly affect plant

W
productivity (Ma et al., 2020a).
IE
1.2 Drought Impacts on Sorghum Growth, and Development

Drought stress significantly impairs water uptake by plant roots due to decreased
EV
soil moisture availability. This reduction in water uptake limits transpiration, a critical

process for thermal regulation, nutrient transport, and the maintenance of turgor pressure,
PR

ultimately slowing plant growth and reducing both biomass accumulation and crop yield

(Seleiman et al., 2021). In response to water deficit, plants induce stomatal closure to

conserve water, which concurrently restricts carbon dioxide (CO₂) uptake and suppresses

photosynthetic activity. The consequent decline in photosynthesis limits energy

availability for growth and reproductive processes. Under prolonged drought conditions,

chlorophyll degradation is often observed, leading to premature leaf senescence and

further reductions in photosynthetic capacity (Qiao et al., 2024).

Moreover, drought stress is associated with increased production of reactive

oxygen species (ROS), including hydrogen peroxide (H₂O₂) and superoxide radicals
 4

(O₂⁻), which can cause oxidative damage to lipids, proteins, and nucleic acids (Cruz,

2008; Qiao et al., 2024)). Although plants activate antioxidant defense systems involving

enzymatic and non-enzymatic components to mitigate ROS-induced damage, severe or

prolonged stress frequently overwhelms these defenses, resulting in cellular dysfunction

and reduced productivity (Laxa et al., 2019). To enhance water acquisition under drought,

plants often undergo morphological adaptations such as modifications to root system

architecture, including increased root depth and branching. These changes allow for the

exploitation of deeper soil moisture reserves (Kim et al., 2020).

W
However, the allocation of resources toward root development can compromise

investment in shoot growth, leaf area expansion, and reproductive structures, thereby
IE
negatively affecting overall plant productivity (Muhammad et al., 2021). In S. bicolor,

genotypes exhibiting a greater number of seminal roots and wider xylem vessel diameters
EV

demonstrate enhanced drought tolerance. Furthermore, varieties with longer and finer

roots have been shown to absorb water more efficiently than those with shorter, thicker
PR

roots, indicating that root length may play a more pivotal role than diameter in conferring

drought resilience (Prasad et al., 2021). Although sorghum is renowned for its

adaptability to arid environments, severe drought events can still substantially impair its

physiological function and yield potential (Begna et al., 2022).

1.3 Salinity Impacts on Sorghum Growth and Development

Salinity, like drought, is a major abiotic stressor that significantly constrains crop

growth and productivity (Oliveira et al., 2013). Salinity stress primarily induces an

osmotic imbalance in plants. Elevated salt concentrations in the rhizosphere reduce the

soil water potential, making water uptake difficult even when moisture is physically
 5

present, resulting in a condition referred to as physiological drought (Hasanuzzaman et

al., 2022a). Consequently, plants exhibit reduced water uptake, inhibited growth, and

diminished biomass accumulation (Ahmad et al., 2023)

In addition to osmotic stress, salinity particularly from sodium chloride (NaCl)

leads to the excessive accumulation of toxic ions such as sodium (Na⁺) and chloride (Cl⁻)

in plant tissues. This disrupts cellular ionic homeostasis and impairs key metabolic

pathways (Atta et al., 2023). Sodium ions compete with potassium (K⁺) for uptake,

reducing potassium availability for enzymatic activity and cellular metabolism.

W
Moreover, salinity-induced nutrient imbalances hinder the absorption of essential

elements such as potassium, calcium (Ca²⁺), and magnesium (Mg²⁺), further exacerbating
IE
growth limitations and yield reductions (Chele et al., 2021).

Salinity stress also induces stomatal closure to reduce water loss, which
EV

simultaneously limits CO₂ assimilation and decreases photosynthetic rates. Salt

accumulation in foliar tissues can lead to chloroplast damage and impaired

PR

photosynthetic efficiency, culminating in lower biomass production (Balasubramaniam et

al., 2023a). Furthermore, salinity disrupts hormonal regulation, notably by increasing

abscisic acid (ABA) levels. While ABA promotes adaptive responses such as stomatal

closure and osmotic adjustment, it also inhibits growth by suppressing cell division and

expansion (Bharath et al., 2021; Chen et al., 2022; Chen et al., 2020). Additionally,

salinity can enhance ethylene production, which may intensify stress responses and lead

to premature leaf senescence (Balasubramaniam et al., 2023b).

Soils are typically classified as saline when the electrical conductivity exceeds 4

dS/m (equivalent to approximately 40 mM NaCl) at 25°C and when exchangeable

 6

sodium levels reach or exceed 15% (Kapadia et al., 2022; Sparks, 1995). Sorghum

bicolor is considered moderately salt-tolerant and can endure salinity levels up to 70 mM

NaCl; however, its response to salinity is highly genotype-dependent. For example,

Dehnavi et al. (2024) reported that forage fresh yield decreased by 10–23% under 60 mM

NaCl and by 21–47% under 120 mM NaCl across diverse sorghum genotypes (Dehnavi

et al., 2024). These findings underscore the importance of characterizing genotype-

specific responses to salinity stress for breeding salt-resilient cultivars, which is vital for

sustaining sorghum productivity in saline soils and enhancing food security in stress-

prone regions.

W
1.4 Effects of Drought and Salinity Pressure on Sorghum Defense and Adaptation
IE
Both drought and salinity stress create a complex environment for sorghum plants

(Hossain et al., 2022c) and can influence the expression of genes related to plant defense.
EV
For example, plants under stress may prioritize survival over defense, increasing

vulnerability to additional stressors (Berens et al., 2019; Iqbal et al., 2021). Studies have
PR

demonstrated that abiotic stress conditions, such as drought and salinity, can weaken a

plant's physical defenses, making it more susceptible to biotic stress, such as aphid

infestations. However, certain genotypes may maintain defenses even under water-limited

conditions (Machingura, 2021). Thus, when faced with multiple stressful factors, plants

can experience even more detrimental effects on their growth and development. For

instance, drought and salinity stress can impair how plants respond to diseases,

weakening their defense mechanisms and resulting in stunted growth or increased

pathogen spread within the ecosystem (Nawaz et al., 2023). Under these challenging

environmental conditions, plants often rely on symbiotic relationships with rhizosphere

 7

microbial communities to help mediate their stress response, as these microbes can assist

in modulating the plant defenses under such stressors (Thepbandit et al., 2024).

1.5 Rhizosphere Microbial Influence on Drought and Salinity Stress

The rhizosphere is the narrow region of soil surrounding plant roots, where

dynamic interactions between plants and microbes occur (Pantigoso et al., 2022a).

Rhizosphere microbial communities, particularly bacteria, play a pivotal role in

enhancing plant resilience to abiotic stress. These microorganisms can modulate plant

defense mechanisms, improve nutrient acquisition, and enhance stress tolerance through

W
induced systemic resistance (ISR) and symbiotic associations (Raaijmakers et al., 2009).

In drought and salinity conditions, specific microbial communities become more

IE
prominent in the sorghum rhizosphere, including plant growth-promoting rhizobacteria

(PGPR), and salt-tolerant endophytes (Vurukonda et al., 2016). According to (Zheng et

EV
al., 2024a), drought-stressed Streptomyces parvum isolates enhanced root growth and

influenced plant responses during drought by colonizing roots and the rhizosphere.
PR

Whereas under salinity stress, halotolerant microorganisms such as Arthrobacter,

Azospirillum, Alcaligenes, Bacillus, Burkholderia, Enterobacter, Flavobacterium,

Pseudomonas, and Rhizobium, have been shown to alleviate salt stress in crops

(Asif et al., 2023; Zhang et al., 2019; Zhang et al., 2023b). This highlights the importance

of rhizosphere microbial diversity in alleviating the negative impacts of salinity on plant

growth. Additionally, research has shown that plant genotypes are crucial in selecting and

recruiting specific microbiomes. The interaction between a particular plant genotype and

its associated microbiome is essential for enhancing the plant's fitness, particularly by

mitigating environmental stresses (Pantigoso et al., 2022b; Yue et al., 2024). However,
 8

modern sorghum genotypes and varieties may not have been optimized to harness the

beneficial effects of naturally occurring microorganisms entirely. This highlights a

significant opportunity to explore microbial communities that can potentially boost

drought and salinity tolerance in sorghum cultivation.

1.6 Drought and Salinity Stress Influence on Rhizosphere Microbial Ecological

Functions

Rhizosphere microbes, particularly those that produce antioxidant enzymes or

stimulate the plant's antioxidant defenses, can mitigate this oxidative damage and

improve the plant's overall resilience (Ngumbi et al., 2016). Salinity and drought harm

W
soil microorganisms by decreasing soil enzyme activity and restricting the cycling of

nutrients in soil ecosystems (such as C, N, and P). Additionally, drought and salinity
IE
stressors drastically affect the rhizosphere's function in nutrient cycling by changing the
EV
makeup of the microbial population and root exudation patterns (Parasar et al., 2024).

Plants frequently alter their exudates during drought to draw microorganisms that

improve nutrient intake and water retention, such as those that produce osmoprotectants
PR

(Nayloret al., 2018a). However, this shift may come at the cost of nutrient availability, as

stressed plants and microbes compete for limited resources (Kumar et al., 2022). Like

salinity stress affects nutrient cycling, excessive salt concentrations can suppress

microbial activity, particularly that of phosphate-solubilizing and nitrogen-fixing bacteria,

which lowers the availability of vital minerals like phosphorus and nitrogen (Shrivastava

et al., 2015a). Salt-tolerant microbial strains may thrive but often contribute less to

nutrient cycling than their non-tolerant microbes (Yang et al., 2021). These shifts

emphasize the complex trade-offs in the rhizosphere under stress conditions, where

microbial communities must balance between promoting plant stress resilience and
 9

maintaining nutrient availability, affecting plant growth and productivity (Chaparro et al.,

2014a).

1.7 Trade-offs in Sorghum Rhizosphere Responses under drought and salinity stress
Trade-offs between defensive systems and biomass production are common

characteristics of sorghum's response to stress (Tuller et al., 2018; Zheng et al., 2024b).

Research shows that under abiotic conditions, sorghum genotypes with robust microbial-

mediated responses may exhibit a change in growth rates (Qi et al., 2022). This dynamic

creates a competitive environment within the plant's metabolism, where nutrients and

W
energy are diverted toward sustaining growth or bolstering defense against abiotic

challenges (Wu et al., 2021). For instance, abiotic-stress resistant genotypes may more
IE
effectively trigger stress responses mediated by the rhizosphere and enhance biomass

growth and accumulation (Demirel et al., 2020) or decrease yield (Pires et al., 2020).
EV
Therefore, trade-off management mediated by the rhizosphere is crucial for enhancing

sorghum productivity under several stresses (Dwivedi et al., 2021; He et al., 2022;
PR

Sivasakthi et al., 2024a). According to recent studies, modifying soil microbial

communities to favour beneficial species or rhizosphere engineering may help alleviate

these trade-offs in plant productivity (Sivasakthi et al., 2024b). Thus, sorghum's

performance under drought and salinity stress could be improved by enhancing microbial

diversity and introducing microbes that promote stress tolerance without hindering

growth. However, to achieve this, further research is needed to determine which

microbial species and metabolites are most beneficial for balancing these trade-offs in

sorghum (Chaparro et al., 2014b).

 10

1.8 Potential for Microbiome-Based Solutions

Given the growing interest in sustainable agriculture and reducing chemical inputs

(Tudi et al., 2021), there is significant potential for harnessing rhizosphere microbial

communities to improve sorghum's resistance to abiotic and biotic stress (Schlemper et

al., 2017). Developing microbial inoculants that promote abiotic stress tolerance (J. Li et

al., 2022) may reduce the need for chemical pesticides and fertilizers, leading to more

environmentally friendly agricultural practices (Timmusk et al., 2017). In addition, next-

generation sequencing and metagenomics advances have provided valuable insights into

the composition and function of rhizosphere microbial communities under different stress

W
conditions (Berendsen et al., 2018; Soliman et al., 2017). This opens new avenues for
IE
selecting and engineering microbial consortia tailored to specific crops and

environmental conditions, impacting global food security (Nunes et al., 2024).

EV
1.9 Significance of the current study

The rapid growth of the global population presents a significant challenge in

achieving food security and eliminating hunger (Fanzo, 2023). With projections
PR

estimating the world population will reach between 9.4 and 10.1 billion by 2050 and

potentially 12.7 billion by 2100, food production must rise substantially to meet the

increasing demand (Bahar et al., 2020). Addressing this challenge requires

comprehensive strategies, including adopting sustainable agricultural practices and

climate change mitigation efforts (Agboklou et al., 2024; Dijk et al., 2021). Sorghum, a

versatile and resilient crop, offers a promising solution to the food security challenge

(Dunjana et al., 2022). However, changing climate poses a significant threat to sorghum

production. Rhizosphere microbial communities associated with sorghum play a crucial

 11

role in enhancing the plant's resilience to environmental stressors such as drought and

salinity. This study focuses on SC265 and SC1345 sorghum genotypes from the founder

nested association mapping (NAM) population to investigate the role of the rhizosphere

microbiome in modulating sorghum defenses against salinity and drought stress. These

NAM genotypes have been previously identified to be resistant (SC265) and susceptible

(SC1345) to sugarcane aphids (Puri et al., 2023a). Using these lines with known resistant

and susceptible traits to sugarcane aphids will allow us to examine whether their

responses to biotic stress follow a similar pattern in abiotic stress. These findings are

crucial for breeding programs aiming to develop resilient crops, as a genotype highly

W
resistant to biotic stress but susceptible to abiotic stress may not be ideal for improving
IE
overall crop stability in changing environments. To investigate this a greenhouse

experiment combined with a metagenomic approach was conducted to explore these

EV
interactions.

The study's first objective is to assess the impact of drought and salinity on
PR

biomass productivity (stems, roots, and height) in both SC265 and SC1345 sorghum

genotypes. We hypothesize that drought and salinity will have a more pronounced

negative impact on SC1345 lines compared to SC265 genotypes. The second objective

examines the effect of drought and salinity on the rhizosphere microbial composition,

community structure, and potential function in SC1345 and SC265 sorghum genotypes.

We hypothesize that there would be significant differences in the microbial community of

SC1345 and SC265, and SC265 would have higher microbial diversity and functional

potential than SC1345 genotypes. Our third objective seeks to use SC265 and SC1345

bulk soil as a baseline to confirm the role of the rhizosphere in recruiting specific
 12

microbial communities, such as plant growth-promoting rhizobacteria (PGPR) and plant

growth regulators (PGR) during stress adaptation. We hypothesized that the bulk soil

would have higher microbial communities but a decreased number of plant growth-

promoting rhizobacteria (PGPR). This study highlights the potential of leveraging

rhizosphere microbial ecology to enhance sorghum's resilience, further supporting its role

as a sustainable food source amidst global climate challenges.

W
IE
EV
PR
 13

Chapter 2

Abiotic Stress Impacts on Sorghum Genotypes' Morphology Measurements and

Productivity

Abstract

This study investigated the effects of drought and salinity stress on biomass accumulation

in two sorghum genotypes: SC1345 (aphid-susceptible) and SC265 (aphid-resistant).

Plants were subjected to medium and high levels of drought and salinity, and biomass

parameters were assessed to evaluate genotype-specific responses. Aboveground biomass

was calculated as the sum of plant height, dry leaf weight (DLW), and height (H), while

W
belowground biomass was measured using dry root weights (DRW). Results showed that

drought significantly reduced biomass in both genotypes (p < 0.01). SC1345 exhibited
IE
significantly higher aboveground biomass (DLW and height; p < 0.01) and also

maintained greater root weights across treatments (p < 0.05). SC265, however, showed
EV
comparable root weights under salinity stress, suggesting better belowground adjustment.

Additionally, SC1345 and SC265 showed no significant differences between genotypes.

PR

This may reflect a trade-off strategy, where resources are preferentially allocated to root

maintenance under stress. These findings highlight complementary traits in both

genotypes, offering valuable insights for breeding climate-resilient sorghum cultivars.

Keywords: Plant Biomass, sorghum, climate change, food security, Plant breeding
 14

2.1 Introduction

Plant development, growth, productivity, and adaptation to climatic change have

become major concerns and interests for plant breeders and agriculture biotechnology

industries (Benitez-Alfonso et al., 2023; Ma et al., 2020b). Plant biomass, including

leaves, stems, roots, and allocation, are fundamental for crop growth, development, and

productivity and are important measures in global food security (Muscat et al., 2020).

Studies have shown that the proportion of belowground (root) biomass to aboveground

biomass (stems, leaves, and branches) serves as a key indicator of how plants allocate

their biomass (Qi et al., 2019). Typically, 15%–20% of plant biomass in the family

W
(Poaceae) grasses, including rice, wheat, corn, and sorghum, is devoted to roots, whereas
IE
80%–85% is distributed among aboveground organs like leaves and stems (Ordonio et

al., 2016; Qi et al., 2019). However, these traits are particularly sensitive to abiotic
EV
stresses, with drought limiting root elongation, stem growth, and salinity impairing

nutrient balance and leaf expansion (Hassan, 2024; Irving, 2015).

PR

In plant productivity, leaves play a pivotal role in photosynthetic activity,

determining the plant's capacity to convert light energy into chemical energy

(Weraduwage et al., 2015). The stem, as the main axis of the plant, supports leaf

arrangement, facilitates nutrient and water transport, and maintains structural integrity

under environmental stress (Poorter et al., 2012). Plant height influences light capture and

competitive ability. Additionally, taller plants were more likely to have greater leaf areas,

and production was highly correlated with leaf area (Angove et al., 2020). Also, the root

system is critical for water and nutrient uptake, anchorage, and interaction with soil

microbiota, specifically the rhizosphere (Molefe et al., 2023; Sainju et al., 2017). Thus,
 15

the coordinated functioning of these organs underpins plant growth and yield potential,

particularly in challenging environmental stress conditions.

Abiotic stresses, including salinity and drought, are significant challenges to

achieving optimal crop performance, as they severely affect plant physiology,

morphology and metabolic processes (Munns et al., 2008). Stress, which can be viewed

as an energy-costing condition, reduces energy acquisition in plants or redirects plants

from growth to stress defense. These stresses can reduce growth rates, compromise

biomass accumulation, and lead to significant yield losses (H. Zhang et al., 2020a).

W
Therefore, plant resilience to stress, such as sorghum, has been of interest because of its

ability to enhance greater energy allocation toward grain production, which is essential
IE
for global food security (Teferra & Awika, 2019).
EV
Sorghum (Sorghum bicolor L.), a key cereal crop adapted to semi-arid and

environmental stress conditions, is a valuable model for studying plant responses to

abiotic stresses (Tu et al., 2023b). Genotypes of sorghum display variability in stress
PR

tolerance mechanisms, such as deeper and more robust root systems for enhanced water

uptake, and thicker stems for structural integrity for maximizing photosynthetic

efficiency under suboptimal conditions (Alzahrani et al., 2025; Ndlovu et al., 2021).

However, sorghum genotypes morphological and productivity responses remain

underexplored under drought and salinity stress. This study focuses on two sorghum

genotypes SC265 previously identified as resistant to sugarcane aphids and SC1345

identified to be susceptible to sugarcane aphids (Puri et al., 2023b)to evaluate the effects

of drought and salinity on stem width, root weight, leaf weight, plant height, and overall

Reproduced with permission of copyright owner. Further reproduction prohibited without permission.