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Microbiomics and Sustainable
Crop Production
Microbiomics and Sustainable
Crop Production
Mohammad Yaseen Mir

Centre of Research for Development
University of Kashmir
Srinagar, India
Saima Hamid
Department of Environmental Science
University of Kashmir
Srinagar, India
This edition first published 2023
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v
Contents
Preface xi
About the Authors xii
1 Agricultural Microbiomes: Functional and Mechanistic Aspects 1

1.1 Introduction 2
1.2 Model Microbiome–Plant Systems 2
1.2.1 Plant Perception of Microbes 3
1.2.2 Molecular Plant 4
1.2.3 Bacterial Signalling: Quorum Sensing and Symbiosis Factors 5
1.2.4 Hormone Signalling in Microbe–Host Interactions 5
1.2.5 Interactome Network Analysis 7
1.2.6 Transcriptional Regulatory Networks 9
1.2.7 Metabolic Exchanges and Nutrient Competition in the Soil 10
1.2.8 Integrated Multi-omics Modelling 10
1.2.9 From Systems Biology to Crop Protection 11
1.3 Stability, Resilience, and Assembly of Agricultural
Microbiomes 11
1.4 Core Plant Microbiome and Metagenome 13
1.5 Interactions Among the Microbes, Environment, and Management 14
1.5.1 Secondary Metabolism 17
1.5.2 Endophyte–Phytopathogen–Plant Interaction 17
1.5.3 Hopanoid 18
1.5.4 Parasitic Interaction 19
1.5.5 Microbial Community’s Interaction 19
1.5.6 Siderophore 20
1.5.7 Symbiotic Interaction 20
1.6 Microbiome Innovation in Agriculture: Insect Pest Management 21
1.6.1 Manipulation of Insect-Associated Microbiomes for Pest
Management 24
1.6.2 Incompatible Insect Technique (IIT) 25
vi Contents
1.6.3 Paratransgenesis 27
1.6.4 Exploiting the Chemical Inventories of Microbiomes to Develop New
Biopesticides 29
1.6.5 Microbial Insecticides and Plant-Incorporated Protectants 30
1.6.6 Microbial Semiochemicals 33
1.6.7 Combining Microbial-Based Biopesticides with Nanotechnologies 36
1.6.8 Microbial Interventions to Improve Fitness of Mass-Reared Insects
for Autocidal Programmes 37
References 39
2 Engineering and Management of Agricultural Microbiomes

for Improving Crop Health 66
2.1 Why to Modify Plant Microbiome? 67
2.2 Methods for Detecting Endophytes Within the Plant 69
2.2.1 Media for Isolation of Fungal Endophytes 70
2.2.2 Media for Isolation of Bacterial Endophytes 71
2.2.3 Identification of Endophytes 72
2.2.4 Molecular Tools to Identify Endophytes 72
2.2.5 Markers and Primers for Endophyte Identification 73
2.2.6 Techniques to Evaluate Endophyte Distribution in Plants 74
2.2.6.1 Hood and Shew Staining Protocol 74
2.2.6.2 Fluorescent Probes for Localization of Bacterial and Fungal
Endophytes 74
2.2.6.3 ROS Staining to Study Bacterial Endophytes 75
2.2.7 Analysis of Endophyte Diversity 76
2.2.8 Non-Culture Methods 77
2.2.9 Metagenomics and Pyrosequencing 77
2.2.10 Microarray: Gene Chips to Study the Expression and Mechanisms
of Interaction 78
2.3 Engineering of the Plant Microbiome 79
2.3.1 Host-Mediated and Multi-Generation Microbiome Selection 79
2.3.2 Inoculation into the Soil and Rhizosphere 80
2.3.3 Inoculation into Seeds or Seedlings 80
2.3.4 Tissue Atomization 81
2.3.5 Direct Injection into Tissues or Wounds 82
2.4 In Situ Harnessing of Agricultural Microbiome 82
2.4.1 Recent Advancement in Plant Microbiome Studies 82
2.4.2 Microbial-Based Strategies 84
2.4.3 Biochemical Strategies 84
2.4.4 Molecular Strategies 85
2.5 Future Perspective of Agricultural Microbiome Engineering 86
References 87
Contents vii
3 Approaches and Challenges in Agricultural Microbiome Research 97

3.1 Microbiome Research in the Omics Era 97
3.2 New Efforts and Challenges in Assigning Function
to Microbes 99
3.3 Characterization of Complex Microbial Communities 101
3.4 Advanced Fundamental Research on Microbe–Microbe
and Plant–Microbe Interactions: Bridging the Lab–Field Gap 102
3.4.1 Bridging the Lab–Field Gap 103
3.4.1.1 Limitations on the Experiments Performed in Controlled Conditions‌:
The Lack of Context 103
References 105
4 Perceptive of Rhizosphere Microbiome 111

4.1 Introduction 112
4.2 Multiple Levels of Selection in the Plant Rhizosphere 113
4.2.1 Microbial Experimental Systems and Network Analysis 113
4.2.2 Observing Microbiome Controls over Observed Phenotypes of the Plant
Using -Omics Techniques 114
4.2.3 Genome-Editing Techniques to Uncover Plant Host Controls over
Microbiome Composition and Function 115
4.2.4 Rhizosphere Engineering and Sustainable Agriculture 115
4.2.5 Engineering Plants 116
4.2.6 Case Study 1: Manipulating Rhizosphere pH 118
4.2.7 Case Study 2: Enhancing Organic Anion Efflux from Roots 118
4.2.8 Approach 1: Engineering Metabolic Pathways for Greater Organic
Anion Efflux 119
4.2.9 Approach 2: Engineering Transport Proteins for Greater Organic Anion
Efflux 120
4.2.9.1 ALMT Family 121
4.2.9.2 MATE Family 121
4.2.10 Engineering Microbes 122
4.2.11 Strategic Issues for Strain Development 123
4.2.12 PGPR Activity Is Enhanced in Engineered Strains 123
4.2.13 Recombinant Strains and Rhizosphere Competence 125
4.2.14 Non-Target Effects of Wild-Type and Genetically Engineered
PGPR 126
4.3 Engineering Microbial Populations and Plant–Microbe
Interactions 127
4.4 Emerging Approaches in Rhizoremediation 128
4.4.1 Impact of Rhizosphere Microbiome on Rhizoremediation 129
4.4.2 Current Approaches to Understand the Role of the Microbiome
in Rhizoremediation 130
viii Contents
4.4.3 Metagenomics 131

4.4.4 Metatranscriptomics 132
4.4.5 Metaproteomics 133
4.4.6 Genomics 134
4.5 Heritability of Rhizosphere Microbiome 137
4.6 Future Course of Orientations 139
References 140
5 Microbial Communities in Phyllosphere 154

5.1 Introduction 154
5.2 Diversity of Microbes in Phyllospheric Environment 156
5.2.1 Sources of Microbes Colonizing the Phyllosphere 158
5.2.2 Leaf Characteristics and Environmental Factors Controlling
Phyllosphere Microbiology 159
5.3 Microbial Adaptation to the Phyllosphere 160
5.3.1 Plant Genotype and Phyllosphere Microbiology 162
5.4 Relationship between Phyllosphere Microbial Communities
and Functional Traits of Plants 163
5.5 Metabolic Dynamics of Phyllosphere Microbiota 166
5.6 Impact of Phyllospheric Microorganisms
on Plant–Plant, Plant–Insect, and Plant Atmosphere
Chemical Exchanges 167
5.7 Quorum Sensing in Phyllosphere 169
5.8 Applications for Phyllosphere Microbiology 171
5.8.1 Biocontrol Agents 171
5.8.2 Plant Growth-Promoting Compounds 173
5.8.3 Biopharmaceutical Importance 174
5.8.4 Other Applications 174
5.8.5 Conclusion and Future Prospects 175
References 176
6 Endosphere and Endophyte Communities 193

6.1 Reproduction and Transmission Modes of Microbes 194
6.2 Vertical Transmission 196
6.2.1 Vertical Transfer via Seeds 196
6.2.2 Vertical Transfer via Pollen 199
6.2.3 Horizontal Transmission 200
6.2.3.1 Colonization of Seed and Root via Soil 200
6.2.4 Endophytic Colonization of the Spermosphere 200
6.2.5 Colonization of the Root Endosphere via the Rhizosphere 201
6.2.6 Entry into Aerial Tissues 202
Contents ix
6.2.7 Aerial Dispersal of the Plant Microbiome 202

6.2.8 Endophytic Leaf Colonization via Stomata 204
6.2.9 Floral Transmission of Bacterial Endophytes 205
6.2.10 Endophyte Transmission by Plant-Feeding Insects 206
6.3 Endophyte Genomes and Metagenomes 207
6.3.1 Genome Analysis 207
6.3.2 Multigenome Analysis 207
6.3.4 Advanced Fundamental Research on Microbe Interactions in the
Endosphere 211
6.3.5 Fungal Hyphae as Vehicles for Bacterial Colonization of the
Endosphere 212
6.3.6 Bacterial Intrahyphal Colonization 213
6.4 Bacteria and Fungi in Mixed Biofilms in Plants 213
6.5 Conclusion and Future Perspectives 216
References 216
7 Core Microbiomes: For Sustainable Agroecosystems 240

7.1 Core Microbiome for Agriculture: A Taxonomic and Functional
Aspect 241
7.1.1 Core Microbiome Identification 243
7.1.2 Functional Core Microbiome 243
7.1.3 Conservative Approaches to Core Plant Microbiomes 245
7.2 Core Microorganisms and Priority Effects in Initial Assembly 249
7.2.1 Microbiome Types 249
7.2.2 Priority Effects in Initial Assembly 250
7.2.3 Deploying Core Microorganisms 251
7.2.4 Prioritizing a Core Microbiome over Space 251
7.2.5 Prioritizing a Core Microbiome over Time 253
7.2.6 Neutral Model to Inform Core Taxa That Are Deterministically
Assembled 254
7.3 Informatics of Microbial Networks 255
7.3.1 Microbial Networks 255
7.4 Designing Core Microbiomes 257
7.4.1 Criterion for Nominating Core Microorganisms 258
7.4.1.1 Functional Species Recruitment 258
7.4.1.2 Pathogen/Pest Blocking 259
7.4.1.3 Core Reinforcement 259
7.5 Management of Agroecosystems with Core
Microbiomes 260
7.5.1 Logistics of Core Microbiomes 260
x Contents
7.5.2 Portfolios with Multiple Cores 261

7.5.3 Smart Farming with AI and Robots 263
References 264
Further Reading 271
8 Microbiome Mediated: Stress Alleviation in Agroecosystems 272

8.1 ffect of Biotic and Abiotic Stresses on Plants 273
E
8.1.1 Biotic and Abiotic Stresses 273
8.1.2 Biotic Stress 273
8.1.3 Abiotic Stress 275
8.1.4 Water Stress 275
8.1.5 Transpiration 276
8.1.6 Water Loss 277
8.1.7 Temperature Stress 277
8.1.7.1 Chilling Stress 277
8.1.7.2 Freezing Stress 278
8.1.7.3 Heat Stress 279
8.1.7.4 Low-Oxygen Atmosphere and High-Carbon-Dioxide
Atmosphere 279
8.1.7.5 Low-Oxygen Atmosphere 279
8.1.7.6 High-Carbon-Dioxide Atmosphere 280
8.1.7.7 Ethylene and Nonethylene Volatiles 280
8.1.7.8 Light 281
8.1.7.9 Mechanical Stress 283
8.1.7.10 Oxidative Stress 284
8.1.7.11 Mineral Stress 284
8.2 Molecular and Physiological Responses of Plants Against
Stresses 285
8.2.1 Morpho-Physiological Responses 285
8.2.2 Molecular Responses 286
8.3 Microbiome Mediated Mitigation of Stress Conditions 288
8.3.1 Improved Understanding of a Microbiome Role in Plant Defence
and Immune Systems 290
8.3.2 Cry for Help’ Strategy for the Applied Plant Stress Probiotics 293
8.4 Multi-Omics Strategies to Address Stress Alleviation 293
8.4.1 Genomics 294
8.4.3 Transcriptomics 297
8.4.4 Proteomics 298
8.4.5 Metabolomics 301
References 303
Index 320
xi
Preface
Microbiomes formed by ages of evolution in soils play an important role in sus-

tainability of crop production by enriching soil and alleviating biotic and abiotic
stresses. This diversity is an essential part of the agroecosystems, which are being
pushed to edges by pumping agrochemicals and constant soil disturbances.
Consequently, efficiency of cropping system has been decreasing, aggravated
further by the increased incidence of abiotic stresses due to changes in climatic
patterns. Thus, the sustainability of agriculture is at stake. Understanding
the microbiota inhabiting phyllosphere, endosphere, rhizosphere, and non-
rhizosphere and its utilization could be a sustainable crop production strategy.
With the advent of omics technologies and gene editing, along with increasing
efforts towards transdisciplinary research across various agriculturally relevant
microbiomes, major breakthroughs and microbial-based innovations for agricul-
ture are greatly anticipated. We are currently experiencing a reinvigoration of
microbial biotechnologies that utilize microbial benefits to improve agroecosys-
tem functioning, for example through enhanced soil health, crop vigour, and pest
protection. This book will be immensely helpful to the students of plant biotech-
nology, agricultural sciences, agricultural engineering, and plant sciences at both
undergraduate and postgraduate levels in universities and colleges. Besides, the
book will be quite supportive to researchers who work in the field of plant bio-
technology, plant sciences, and agricultural sciences.
Key features are:
1) Engineering of the plant microbiome
2) Advanced fundamental research on microbe–microbe and plant–microbe
interactions (bridging the lab–field gap)
3) Rhizosphere engineering and sustainable agriculture
4) Multi-omics strategies to address stress alleviation
5) Endosphere and endophyte communities
6) Core microbiomes: for sustainable agroecosystems
xii
About the Authors
Dr. Mohammad Yaseen Mir

Senior Researcher
Centre of Research for Development
University of Kashmir Srinagar-190006
Teacher
Department of School Education, J&K Government, India
Dr. Mohammad Yaseen Mir was working as a senior researcher in a research pro-
ject funded by Ministry of Environment, Forest & Climate Change (MoEF & CC),
Government of India along with G.B. Pant National Institute of Himalayan
Environment & Sustainable Development under National Mission on Himalayan
Studies (NMHS) and has recently joined Department of Education J&K as a
teacher. He did his MPhil and PhD Programmes from the University of Kashmir.
He was awarded Doctorate Merit Scholarship by the University of Kashmir to
pursue his PhD programme. He was awarded certificate of appreciation by the
About the Authors xiii
University of Kashmir for participation in national seminar on ‘Environment

Pollution: Join the Race to Make the World a Better Place’ on the Eve of Observance
of World Environment Day 2016. He earned a PhD in Botany with a specialization
in plant biotechnology on the topic ‘Role of elicitors for in vitro induction of sec-
ondary metabolites in suspension cultures of Artemisia amygdalina D’ from the
University of Kashmir, India. He is currently editor and reviewer of many reputed
journals. Dr. Mir is also subject course expert for environmental science approved
CEC-Moocs by MHRD GoI, New Delhi. He has published many research articles,
review papers, book chapters in reputed, referred international and national jour-
nals like Springer/Elsevier/Hindwaii/Frontiers. He has also authored and edited
many books like Sustainable Agriculture: Biotechniques in Plant Biology with
Springer, Nano-Technological Intervention in Agricultural Productivity with John
Wiley, and Core Microbiome: Improving Crop Quality and Productivity with
John Wiley.
Dr. Saima Hamid

Senior Research Fellow
Department of Environmental Science
University of Kashmir Srinagar-190006, India
Dr. Saima Hamid holds Doctorate as well as Master’s Degree in Environmental

Science from the University of Kashmir, India. She has qualified UGC-NET exams
multiple times and other state level exams. Her research area includes Plant
molecular biology, stress adaptations, bioactive compound isolation and charac-
terization, climate change and microbial biotechnology. She has published more
than thirty research and review articles in highly reputed journals like Elsevier,
Frontier’s in Plant Sciences, Springer, MDPI, Wiley and other peer reviewed
journals. She is the Co-founder of CERD Foundation, member of various inter-
national organisations which focuses on Plant adaptations and climate change.
xiv About the Authors
She is an international forum speaker from six years and columnist of various
international magazines and local newsletters in different countries like Island
Chief of Maldives, Sri Lanka, Bangladesh and many others.
Recent Publication
Din, S., Hamid, S., Yaseen, A. et al. (2022). Isolation and Characterization of
Flavonoid Naringenin and Evaluation of Cytotoxic and Biological Efficacy of
Water Lilly (Nymphaea mexicana Zucc.). Plants. 11, 3588. https://doi.org/10.3390/
plants11243588
Hamid, S. and Mir, M.Y. (2021). Global Agri-Food Sector: Challenges and
Opportunities in COVID-19 Pandemic. Front. Sociol. 6:647337. doi: 10.3389/
fsoc.2021.647337
Hamid, S., Mir, M.Y., Rohela, G.K. (2020). Novel coronavirus disease (COVID-19): a
pandemic (epidemiology, pathogenesis and potential therapeutics). New Microbes
New Infect. Apr 14;35:100679.
Sahu, P.K., Jayalakshmi, K., Tilgam, J. et al. (2022). ROS generated from biotic stress:
Effects on plants and alleviation by endophytic microbes. Front. Plant Sci.
13:1042936. doi: 10.3389/fpls.2022.1042936
Sahu, P.K., Tilgam, J., Gupta, A. et al. (2022). Surface sterilization for isolation of
endophytes: Ensuring what (not) to grow. Basics of microbiology, 62 (6), 647–668.
1
Agricultural Microbiomes
Functional and Mechanistic Aspects
CONTENTS
1.1 Introduction, 2
1.2 Model Microbiome–Plant Systems, 2
1.2.1 Plant Perception of Microbes, 3
1.2.2 Molecular Plant, 4
1.2.3 Bacterial Signalling: Quorum Sensing and Symbiosis Factors, 5
1.2.4 Hormone Signalling in Microbe–Host Interactions, 5
1.2.5 Interactome Network Analysis, 7
1.2.6 Transcriptional Regulatory Networks, 9
1.2.7 Metabolic Exchanges and Nutrient Competition in the Soil, 10
1.2.8 Integrated Multi-omics Modelling, 10
1.2.9 From Systems Biology to Crop Protection, 11
1.3 Stability, Resilience, and Assembly of Agricultural Microbiomes, 11
1.4 Core Plant Microbiome and Metagenome, 13
1.5 Interactions Among the Microbes, Environment, and Management, 14
1.5.1 Secondary Metabolism, 17
1.5.2 Endophyte–Phytopathogen–Plant Interaction, 17
1.5.3 Hopanoid, 18
1.5.4 Parasitic Interaction, 19
1.5.5 Microbial Community’s Interaction, 19
1.5.6 Siderophore, 20
1.5.7 Symbiotic Interaction, 20
1.6 Microbiome Innovation in Agriculture: Insect Pest Management, 21
1.6.1 Manipulation of Insect-Associated Microbiomes for Pest Management, 24
1.6.2 Incompatible Insect Technique (IIT), 25
1.6.3 Paratransgenesis, 27
1.6.4 Exploiting the Chemical Inventories of Microbiomes to Develop New
Biopesticides, 29
1.6.5 Microbial Insecticides and Plant-Incorporated Protectants, 30
1.6.6 Microbial Semiochemicals, 33
Microbiomics and Sustainable Crop Production, First Edition. Mohammad Yaseen Mir
and Saima Hamid.
© 2023 John Wiley & Sons Ltd. Published 2023 by John Wiley & Sons Ltd.
2 1 Agricultural Microbiomes
1.6.7 Combining Microbial-Based Biopesticides with Nanotechnologies, 36

1.6.8 Microbial Interventions to Improve Fitness of Mass-Reared Insects for
Autocidal Programmes, 37
References, 39
1.1 Introduction
In recent years, the microbial environment has gotten a lot of attention because
lower sequencing costs have allowed for more in-depth study of the structure and
dynamics of host-associated microbiota. It is widely acknowledged that microbes
have immense ability to improve host well-being in both humans and plants.
Targeted application of beneficial microbial cocktails can be a sustainable way to
mitigate biotic and abiotic stress conditions and maintain yield stability in the
potential vision of precision agriculture. Many beneficial microbes, on the other
hand, have similar pathogenic relatives, and it is unknown how the plant immune
system distinguishes between pathogenic and beneficial microbes in order to
combat infection by the former and promote colonization by the latter. It is pos-
sible that even the earliest eukaryotes were overwhelmed by a variety of prokary-
otes, and that eukaryotic immune systems developed to distinguish between
beneficial and pathogenic bacteria. As a result, a deep and complex interaction
between microbes and hosts is predicted, affecting every aspect of eukaryote biol-
ogy. Traditional as well as systems biological ‘omics’ and computational model-
ling methods would be needed to understand microbe–host interactions.
1.2 Model Microbiome–Plant Systems
Plant microbiome researchers must design new model systems as well as draw on
existing systems to integrate microorganism populations as an emerging group. To
create culture collections, large-scale microbial isolation activities and genome
sequencing programmes will be required, as will concerted group efforts to build a
set of uniform protocols and growth platforms. A small flowering angiosperm in the
mustard family, Arabidopsis thaliana, is an example of a popular, albeit non-
agricultural, model for plant microbiome study. The Arabidopsis scheme, however,
has shortcomings, including a lack of symbiotic relationships with nodulating
nitrogen-fixers and mycorrhizal fungi, as well as genomic and phenotypic variations
from essential monocot crops. As a result, there would be a need for multiple model
systems. To figure out which processes can transfer to crops at different evolutionary
distances, a collection of model plant species is needed [1]. The legumes Medicago [2],
Populus [3], rice [4], Sorghum [5], Miscanthus [6], maize [7], and tomato [8] have all
1.2 Model Microbiome–Plant System 3
made progress towards the development of model host–microbiome systems. Many

of these model organisms have completely sequenced genomes and burgeoning sci-
entific populations, allowing them to be used in more microbiome studies. The com-
plete maturation of model systems necessitates coordinated initiatives to create
public services, such as repositories and curated databases for sequenced culture col-
lections of related microbiota. A multidisciplinary group of academic and commer-
cial plant microbiome scientists, with funding from funding agencies, is needed to
develop successful model systems for elucidating plant–microbiome interactions
with full interoperability – a key move towards building a knowledge base for long-
term high-yielding agriculture innovation.
1.2.1 Plant Perception of Microbes

Pathogens and endophytes must first resolve systemic obstacles such as cell
walls [9], waxy epidermal cuticles [10], and constitutive antimicrobial compounds
like phytoanticipins. The evolutionary proximity of beneficials and pathogens can
be explained in part by this common criterion. Plant surface receptors called
pattern-recognition receptors (PRRs) detect the presence of microbes close to the
cell membrane [11]. As per findings of Boller and Felix [12] and Macho and
Zipfel [13], in the defence mechanism of pathogen- or microbe-triggered immu-
nity (PTI/MTI), the intracellular signalling helps in culmination of conserved
pathogen-or microbe-associated molecular patterns (PAMPs/MAMPs); for exam-
ple bacterial flagellin or elongation factor thermo unstable (EF-Tu) as MTI mech-
anism embrace production of reactive oxygen species (ROS), nitrogen oxide
species (NOx), nutrient allocation, metabolite release against microbial protec-
tion, and release signalling molecules for defence purposes and other functions,
including transcriptional modifications. Beneficials and pathogens, on the other
hand, have common, if not identical, molecular patterns, making separation by
unique PRRs impossible. FLS2, which recognizes flg22, the most conserved motif
in bacterial flagellin, is one of the most important models for studying PRR func-
tion [14]. BAK1 is a coreceptor that FLS2 has to activate downstream signalling.
BAK1 is also a coreceptor for BRI1, a leucine-rich repeat receptor kinase (LRR-RK)
that detects plant brassinosteroids (BR) and serves as an integrator between
defence and growth signalling [15]. Certain portions of the protein are recognized
by other receptors. Tomato can sense flgll-28 in an FLS2-independent manner by
FLS3 [16], and the rice pathogen Acidovorax avenae has a separate flagellin motif,
CD2-1, whose receptor is still unknown [17]. Interestingly, some A. avenae strains
are resistant to flagellin glycosylation detection [18]. In comparison to viruses,
certain beneficials have epitopes that resist detection by either or both recep-
tors [19]. Apart from MAMP-masking or avoidance processes, certain beneficials
are possibly identified by their flagellin and inhibit full-fledged immune responses
by yet unexplained mechanisms. Most genes caused by experience of distilled

flg22 in Arabidopsis were downregulated in response to colonization by the com-
mensal Rhizobium sp. 129E [20]. According to their findings, this commensal is
capable of interfering with MAMP-induced transcriptional responses through
alternative pathways. Since this Rhizobium strain lacks the type III secretion sys-
tem (T3SS) or Nod factor biosynthesis genes, signalling from other heteromeric
PRR complexes is likely to play a role.
1.2.2 Molecular Plant

Interactions between symbionts and plants point to pathways that underpin the
distinction between friend and foe. When arbuscular mycorrhizal fungi (AMF)
and rhizobia come into contact for the first time, they cause temporary defence-
like responses that are easily suppressed [21]. Signalling by the Myc and Nod fac-
tors is thought to be essential in this repression [22]. Both symbiotic signals are
characterized by their ability to induce nuclear calcium oscillations through a sig-
nalling cascade involving many conserved symbiotic proteins [23]. Lysine motif
(LysM) receptors, like kinases (RLK), detect Nod factors in hosts, and it is thought
that related receptors exist for Myc factors [24]. Any of these receptors tend to be
involved in pathogen identification. OsCERK1 is a LysM-RLK that is needed for
the establishment of mycorrhizal root symbiosis as well as resistance to rice blast
fungus [25], indicating that it functions as a ‘molecular transition’ between sym-
biotic and defence responses. According to Gourion et al. [22] the exact function-
ing is not clear, although LysM-RLK is found to be of some specific mechanism
with frequent occurrence. NFP is a Nod factor receptor found in Medicago trunca-
tula that regulates vision and defence against the fungus Colletotrichum trifolii, as
well as the oomycetes Aphanomyces euteiches and Phytophthora palmivora [26,
27]. Combinatorial physical interactions between receptors and coreceptors are
essential for signal specificity and integration, according to detailed studies of
exemplary PRRs and LysM-RLK. In nature, plant roots come into contact with a
slew of MAMPs as well as a smorgasbord of signalling molecules. As a result, it is
conceivable, if not likely, that a personalized response to complex microbial
assemblages is installed through a network of interacting receptors’ combinato-
rial and quantitative perceptions of the various signalling molecules. As a result,
PRR signalling would include the use of interconnected global systems approaches.
Smakowska-Luzan et al. [28] performed a proteome-scale interactome analysis,
which is a significant step towards a complete understanding of this important
plant perception mechanism. Also, 225 LRR-RKs (CSILRR) formed a physical cell
surface interaction network in A. thaliana, which they mapped using biochemical
pull-down experiments. CSILRR showed that all LRR-RKs are highly intercon-
nected, clustering into many modules of unknown biological significance.
Importantly, the authors demonstrated that not only direct connections but also
indirect network effects modulate downstream signalling performance, and that
the entire network contributes to the plant immune system’s well-balanced
responses. Understanding plant immunity would need a better understanding of
the LRR-RK network’s automated information processing.
1.2.3 Bacterial Signalling: Quorum Sensing and Symbiosis Factors

Plants use metabolites, volatiles, symbiosis cues, and quorum sensing (QS) mole-
cules to detect bacterial contact in addition to sensing conserved microbial pat-
terns [29]. N-Acyl homoserine lactones (AHL) are important components of
bacterial contact that plants can detect. This was shown with the beneficial
Acidovorax radicis N35, where the AHL-producing wild form was able to dampen
the barley defence response, while flavonoid defence was unregulated after inocu-
lation with the non-AHL-producing mutant [30]. Other examples demonstrate
the growth-promoting and priming effects of AHLs on host plants such as
Medicago, tomato, Arabidopsis, and barley [31].
According to Cha et al. [32] and von Bodman et al. [33], since pathogenic bac-
teria often manufacture AHL, these signalling substances are unlikely to provide
enough information for the plant to modulate its defensive responses on their
own. It is possible that the QS molecule compositions and concentrations suggest
an unbalanced microbial composition. Although the biochemical effects of AHLs
have been studied in depth, the processes and mechanisms by which plants
perceive these bacterial molecules are yet to be discovered [34]. Even in plants
that do not form symbiosis, lipochitooligosaccharides, i.e. Myc and Nod symbiosis
factors, may promote root production, seed germination, and plant growth. As a
result, the detection and signalling mechanism for symbiosis factors is partly
independent of the host’s symbiosis competence. More research is required to
understand how the plant interprets the wide range of rhizosphere signals emitted
by microorganisms, and how different molecules can interact synergistically or
antagonistically to affect plant growth and stress resistance [35].
1.2.4 Hormone Signalling in Microbe–Host Interactions

Signalling by phytohormones is crucial to almost all plant processes. Salicylic
acid (SA), jasmonic acid (JA), and ethylene (ET) are the primary mediators of
defence responses. JA and ET mediate induced systemic resistance (ISR) and
protection against necrotrophs and insects, while SA mediates structural activ-
ity relationships (SAR) and defence against biotrophic and hemibiotrophic
pathogen invasion [36]. Auxin, gibberellins (GA), BR, or cytokinins (CK) are
hormones that primarily regulate developmental processes or abiotic stress
responses (abscisic acid [ABA]). Hormone signalling is heavily integrated, and

multiple hormones control every mechanism of concern, beyond these seem-
ingly clean classifications [37]. Phytohormones are therefore essential for bidi-
rectional communication between plants and microbes. Strigolactones are
exuded from roots in response to phosphate or nitrogen deficiency in order to
attract AM fungi, and their biosynthesis is downregulated after the fungus has
colonized [38]. GA, SA, and ET, on the other hand, inhibit both AM and root
nodule symbiosis, while auxin and ABA promote AM production in a
concentration-dependent manner. For nodule forming, CK and localized auxin
signalling are necessary. Depending on the circumstances and plant organisms,
the position of JA in symbiosis establishment is uncertain and can be positive,
negative, or neutral. Beneficial and pathogenic bacteria also influence the hor-
mone signalling pathway. Coronatine (COR) is a toxin developed by the patho-
genic Pseudomonas syringae pv. tomato DC3000 (Pst) that is similar to plant
JA-isoleucine (JA-Ile) but is more active [39]. The required SA-mediated
defences against the hemibiotrophic Pst are suppressed as a result of the activa-
tion of JA-dependent defence mechanisms [40]. Pathogens control plant signal-
ling in general to inhibit defensive responses and redirect nutrients to infested
tissues for long-term pathogenic colonization [41]. According to Singh et al. [42]
and Li et al. [43], presence of beneficial strains including Pseudomonas fluores-
cens Pf4, Pseudomonas aeruginosa Pag, or Bacillus velezensis LJ02 enhances
endogenous SA levels while some strains are proved to show opposed results in
A. thaliana as well as decreasing JA-Ile levels as reported by Srivastava et al. [44];
however, Paraburkholderia phytofirmans PsJN decreases expression of JA-
biosynthesis and wound-induced JA accumulation [45]. As a result, phytohor-
mones derived from microbes have a wide range of effects, based on the
plant–microbe mix. While various studies show contradictory findings, the SA
signalling mechanism appears to be important for shaping the root microbiome.
SA mutants had mild effects on microbiome structure, according to one
report [46]. On the other hand, according to Lebeis et al. [47] the rhizosphere
microbiota of A. thaliana mutants lacking SA synthesis or vision was altered,
whereas the corresponding JA and ET mutants had no such impact. Many plant-
growth promoting rhizobacteria (PGPRs) influence plant development, espe-
cially root growth, by producing auxins, GA, or CK, in addition to influencing
defences, which is common to pathogens and beneficials. To unravel the under-
lying complexity, systems biology tools such as metabolomics, global network
analysis, hormone profiling, and specialized quantitative modelling of molecu-
lar processes in plants and soil will be necessary. Auxin signalling in the plant
root is being extensively investigated, and sophisticated models are availa-
ble [48]. Detailed mechanistic information as well as fluorescent auxin reporters
that offer time-resolved data on auxin distribution permitted the construction of
such quantitative models [49]. Quantitative time-resolved models are built on

the foundation of both. Although preliminary evidence on the effects of auxin
concentrations on receptor pairs is available, quantitative data on the chemicals
and receptors that convert a given auxin concentration into specific transcrip-
tional responses are often lacking [50]. A model of the SA signalling system will
be useful in studying microbe–host interactions. NPR1, NPR3, and NPR4 are
newly discovered SA receptors that work together to modulate responses to vari-
ous SA concentrations [51]. BOP1 and BOP2, on the other hand, appear to have
no role in SA signalling but have been linked to developmental programmes in
legumes such as blooming and nodule formation [52]. Simultaneously, the bio-
chemical control of NPR1 and presumably its paralogs is complicated, including
several cellular compartments, redox potential, phosphorylation, and degrada-
tion. Although critical aspects for model creation such as thermogravimetric
analysis (TGA) transcription factors and signalling network components are
known [53], our knowledge of this important immune signalling system is still
limited. For quantitative modelling of SA signalling, the creation of fluorescent
SA sensors as well as quantitative protein level and binding data are crucial. All
hormone signalling pathways are linked, and only a few biological reactions are
mediated by a single hormone. The integrative research of Tsuda et al. [54] con-
stitutes great efforts in unravelling the interaction of SA, JA, and ET during
immunity in Arabidopsis. They separated the hormone signalling network into
four sectors (SA, JA, ET, and PAD4) and tested immunity in all potential mutants
from each sector following stimulation with a panel of MAMPs and effectors.
Their findings revealed substantial hormone network component interactions,
including additive, synergistic, and compensatory interactions. Later research
by the same group led to the conclusion that the PTI signalling network is
strongly buffered against interference, such as pathogen effectors [55].
1.2.5 Interactome Network Analysis

Molecular interaction network techniques can be useful in the absence of quanti-
tative dynamic models for identifying modules, routes, components, and system-
level patterns of molecular host–microbe interactions [56]. A reference protein
network is necessary to situate host–microbe interaction data in the context of
host biology. With the publishing of the first experimental map of physical
protein–protein interactions among several thousand Arabidopsis proteins, plant
interactome analysis began:
Arabidopsis Interactome-1 (AI-1) [57] offered a first integrated organizational
view of plant molecular connectivity. Since then, numerous specialized and com-
plementary maps have been developed to aid in the investigation of certain pro-
cesses. Using the split-ubiquitin technique, a map of roughly 12 000 protein–protein
interactions was created for membrane proteins [58]. A G-protein interactome

showed a novel role for G-proteins in the regulation of cell-wall modification, a
critical defensive mechanism [59]. Interolog mapping was used to create a
protein–protein interaction network for the fungus Phomopsis longicolla, which
causes Phomopsis seed damage in soybean [60]. To control plant defence and
physiology, pathogens and beneficial microorganisms can release hundreds of
(virulence) effector proteins into the cytosol and apoplast of the host plant [12]. To
fully appreciate host–microbe interactions, their functions must be compre-
hended in a holistic and time-resolved manner. Small-scale investigations of
plant-targeted pathogen effectors revealed that virulence effectors affect host pro-
tein activities to interfere with immune responses and cause illness, a phenome-
non known as effector-triggered susceptibility (ETS) [61]. Effector-triggered
immunity (ETI) is initiated when a pathogen’s effectors are recognized by a host
resistance protein (R protein) [61]. A large-scale interactome study (PPIN-1)
mapped the interactions of virulence effectors from the bacterial pathogen Pst
and the oomycete pathogen Hyaloperonospora arabidopsidis with proteins in the
AI-1 host network to get a systems-level perspective on effector functions [62]; a
follow-up study later added interactions of effectors from the biotrophic ascomy-
cete Golovinomyces orontii [63]. The findings demonstrated that pathogen effec-
tors partially converge on common host proteins, many of which are highly linked
hubs in the host network. The host proteins showed genetic validation rates rang-
ing from 100% for the most strongly targeted proteins to 40% for the less highly
targeted proteins, depending on the degree of convergence. In addition to conver-
gence, numerous effectors targeted proteins throughout the host network, most
likely as a result of the immunological signalling network’s high buffering [55].
Positive and balanced selection was seen in the immediate network neighbour-
hood of the highly targeted proteins, according to population genetic analysis. As
a result, pathogen-induced selection pressure appears to be absorbed by the net-
work that surrounds the effector targets [63]. These data support the idea that
host–microbe interactions are mediated by a complex network that can only be
partially comprehended by looking at individual routes. Pathogens appear to alter
host networks rather than deconstructing network integrity, according to research
on the Yersinia pestis interactome [64]. Pathogens are not the only ones that pro-
duce effector proteins. Plant immune responses and symbiotic relations can be
modulated by mycorrhizal fungi, endophytic fungi, and nitrogen-fixing rhizobia
effector proteins [65]. Many PGPRs, such as Pseudomonas simiae WCS417, and
many proteobacterial strains are anticipated to contain functioning T3SS and
effectors in complex microbiome data sets [66]. It is recognized that virulence
effectors are necessary for productive and beneficial interactions between the ben-
eficial fungus Serendipita indica and rhizobial bacteria. Bradyrhizobium elkanii
T3SS-delivered effectors even allowed soybean nodulation without the need for
Nod factor [67]. Many proteobacteria contain type IV and type VI secretion
systems, which may transfer bacterial protein into hosts and other microorgan-
isms in addition to T3SS. P. simiae WCS417 possesses two T6SS loci [66] and may
send effectors to both its plant host and other competing microorganisms to alter
the microbiota. Proteomic techniques can help researchers better understand the
variety of bacteria effector repertoire [68]. In a study comparing the genomes of a
beneficial soil fungus, Colletotrichum tofieldiae, and a closely related pathogenic
counterpart, Colletotrichum incanum, researchers discovered that while their
secretomes were similar, the beneficial fungus had 50% fewer effector genes and
lower activation of pathogenicity-related genes in plants [69]. As a result, micro-
bial secretomes, as well as the quantity and kind of secreted effectors, may serve
as a key point of distinction between beneficials and pathogens. Non-pathogenic
interactions are most likely influenced by the beneficial effectors’ complement.
Understanding the global dynamics of effectors targeting different areas of the
host network, and how this dynamic connects to ETS and ETI, as well as the
systems-level and dynamic variations between pathogen and helpful effectors’
secretion, will be a major issue for systems biology. RNA, which is transported to
the host through extracellular vesicles, has emerged in recent years as a key com-
munication molecule between hosts and microorganisms (EVs). EVs were ini-
tially discovered in mammalian cells and are now found in bacteria, archaea, and
eukaryotes. In Arabidopsis, small RNA from the fungus Botrytis cinerea was
shown to target host defence genes [70]. Plant EVs and multivesicular bodies
accumulate around plasmodesmata during fungal infections to facilitate callose
deposition at infection sites via host-induced gene silencing (HIGS) using
dsRNA. Another layer of communication is formed by EVs and their RNA pay-
load, whose relevance is just becoming apparent [71].
1.2.6 Transcriptional Regulatory Networks

Transcriptional profiling is frequently utilized, and the findings of significant
research are referenced throughout this article. While comparative transcriptomics is
prevalent, causal regulatory networks and co-expression correlation networks are
less so. Co-expression networks are based on the idea that time series transcript pro-
files might reveal causal links between transcripts. Weighted Gene Correlation
Network Analysis (WGCNA) is a widely used approach for grouping genes into co-
expression modules using hierarchical clustering [72]. Signalling network connec-
tions, metabolic pathways, and phenotypic features are all used to compare these
modules. Saelens et al. [73] examined 42 alternative clustering, decomposition,
biclustering, and iterative network inference approaches in addition to WGCNA. In
Arabidopsis, these methods were used. Kim et al. [74] used A. thaliana and other
plants including maize and wheat to study their interactions with microorganisms.
The discovered modules give a preliminary look at genes with similar functions and
can aid in the understanding of processes related to infection or commensalism.
1.2.7 Metabolic Exchanges and Nutrient Competition in the Soil

Metabolic exchanges are one of the core concepts of microbiome–host interactions.
Plant roots deliver up to 40% of the complex carbons generated by photosynthesis to
the rhizosphere, nourishing the microbiome [75]. Fungi and bacteria, on the other
hand, aid in the solubilization and absorption of critical elements like phosphorus,
nitrogen, and iron by the plant [76]. Plant reprogramming by pathogens via effectors
and hormone signalling aims to relocalize nutrients as the metabolism of an indi-
vidual organism has been studied using genome-scale metabolic modelling, and
community-level response modelling is in progress but difficult [77]. Metabolic mod-
elling of prokaryotes is increasingly commonplace [78]; metabolic models for
Arabidopsis, barley, maize, sorghum, sugarcane, and canola have been developed on
the plant side. By comparing metabolic capacities of beneficials and pathogens, the
metabolic capacities of beneficials and pathogens may be assessed [79]. P. syringae
has evolved to be metabolically adapted for a plant-pathogenic lifestyle, according to
Mithani et al. [80]. The pathogenic P. syringae is metabolically extremely similar to its
benign cousin P. fluorescens Pf-5, according to a comparison of metabolic networks
for nine Pseudomonas strains, suggesting that metabolism may not be a crucial dif-
ferentiating trait. A genome-scale metabolic model for the oomycete Phytophthora
infestans was recently created, which predicts biochemical processes in various cel-
lular compartments and in the context of the pathogen’s stage [81]. To provide a
more exact picture of the metabolic changes caused in plant and microbe during
colonization, these models will need to be constrained by metabolite levels.
1.2.8 Integrated Multi-omics Modelling

Although reciprocal benefit between plants and their microbiome is evident, and
a ‘cry for assistance’ might recruit bacteria to aid the host, it is unknown how the
plant combines microbial identification with nutrient-related signals at this time.
Phosphorus is frequently abundant in soil, while plant-absorbable orthophos-
phate is rare [82]. Castrillo et al. [83] explored the relationship between diet and
defence in a stunning multi-omics systems biology exercise. They demonstrated
that the plant phosphate starvation response (PSR) plays a crucial role in modify-
ing the root microbiome by combining 16S rRNA sequencing, genome-wide
expression analysis, analysis and modelling of SynComs, and functional experi-
ments. When phosphate uptake-deficient and phosphate hyperaccumulating
Arabidopsis mutants were compared to wild type, they found that they constructed
distinct root-associated microbiomes. The transcription factors PHR1, and
1.3 Stability, Resilience, and Assembly of Agricultural Microbiomes 11
probably PHL1, are integrators of PSR and immune responses, as phr1 and phr1;
phl1 mutant plants were more resistant to the oomycete and bacterial pathogens.
The relationship between PSR and plant immunity appears to be influenced not
only by the surrounding microbiota but also by pathogens, bringing new issues
concerning the distinctions between helpful and pathogenic bacteria [84].
1.2.9 From Systems Biology to Crop Protection

Understanding crop–microbe connections is becoming easier because of concep-
tual and molecular advancements in microbe–host biology. McGrann et al. [85]
utilized a draught genome assembly to estimate a secretome of roughly 1000 pro-
teins in the developing foliar fungal barley pathogen Ramularia collocygni, which
causes Ramularia leaf spot. They postulated that R. collocygni first acts as an endo-
phyte without generating disease symptoms before transitioning to a necrotrophic
phase, based on the reduced amount of plant cell-wall-degrading enzymes and
the presence of genes associated with chitin recognition avoidance. Systems bio-
logical analysis will rely heavily on understanding such dynamics as well as the
underlying molecular processes and signals. The host specialization of four
Rhynchosporium species on grasses was studied in another research [85].
Rhynchosporia are hemibiotrophic fungi that invade the intercellular matrix of
host plants gradually and without causing symptoms. Six unique effector proteins
from Rhynchosporium commune were revealed to be important for maintaining
the biotrophic growth stage in favour of the necrotrophic destructive stage, giving
therapy leads. Beneficial microorganisms’ impacts on enhanced biomass and
greater tolerance to biotic and abiotic stressors in monocot crops were explored in
a groundbreaking study that included multi-omics techniques. Using phenotyp-
ing, transcriptomic, molecular, and metabolomic techniques, Fiorilli et al. [86]
investigated the three-way interactions between Xanthomonas translucens, the
protective symbiotic AM fungus, and the host. They proposed a two-step process
for conferring Xanthomonas resistance to AM-treated wheat: first, the activation
of a broad-spectrum defence (BSD) response in the roots and leaves of AM-treated
plants, and, second, a switch to pathogen-specific defence (PSD) upon bacterial
infection, which ultimately leads to pathogen resistance.
1.3 Stability, Resilience, and Assembly of Agricultural

Microbiomes
Knowing the identification and functional features of microorganisms present in

crop plant ‘core’ microbiomes is important, but understanding the processes by
which microorganisms assemble into such communities is crucial for any attempt
to control or regulate the agricultural microbiome. The tremendous diversity of

plant microbial communities creates a formidable barrier to properly compre-
hending the ecology of plant-associated microbiomes, and as a result, the relation-
ships between microbiome assembly and plant performance remain little
known [87]. As a result, we urge that researchers focus their efforts on developing
synthetic microbial communities that can colonize plant organs and remain long
enough in natural conditions to provide advantages to the host. What characteris-
tics of a synthetic microbial population make it more likely to colonize plant
organs is a fundamental topic. Microbial genes that are consistently represented
in core metagenomes – and that are enriched in plant microbiomes when com-
pared to soil – are promising candidates for important activities that boost coloni-
zation capacity. Community characteristics such as phylogenetic diversity and
species richness may also influence colonization success, and these might be
investigated in the lab utilizing gnotobiotic model systems and culture collec-
tions [88]. What qualities enable a synthetic community to survive invasion and
displacement by plentiful microorganisms in the surrounding environment once
it has colonized a plant and what characteristics provide resistance to abiotic
stresses, or at the very least the capacity to recover from them, both are question-
able. These traits might be the same as or different from the ones that allowed the
colonization to begin in the first place. All of these issues might be investigated in
the lab using gnotobiotic systems before being tested in greenhouse and outdoor
studies. Years of agronomic work refining single-strain distribution via seed coat-
ings, clay particles, and peat have shown that, in addition to genetic features of the
microbiome, the technique of microbiome inoculation may play a large role in its
effectiveness. Seeding numerous described microbial species into resilient parti-
cles for distribution by air, water, soil, or new delivery mechanisms such as insect
vectors might be used to develop successful microbiome inoculation tactics for
agriculture. However, attempts to increase colonization ability should also guar-
antee that synthetic communities do not overrun local ecosystems and have a
detrimental impact on soil health, nearby plants, or future crops [89]. Finally,
what external variables influence a positive synthetic microbiome’s success?
Synthetic communities may need to be adaptable to changes in host phenotypic
features that affect assembly, or they may need to be customized to colonize cer-
tain crop species. Similarly, the environment will influence a community’s ability
to colonize or its resilience. Any attempt to regulate agricultural microbiomes
would require an understanding of both abiotic (e.g. temperature, light, acidity,
nutrient, and water availability) and biotic (e.g. competition, predation, parasit-
ism, and mutualism) factors within the microbiome elements impacting its
assembly [89]. It will be especially challenging to test the impact of biotic factors
on microorganisms that cannot be cultivated independently. Strong interactions,
for example, can ‘link’ organisms to other microorganisms (e.g. mycoparasitism).
1.4 Core Plant Microbiome and Metagenom 13
Network analysis is particularly beneficial for discovering related microorganisms

and ‘hub’ microorganisms, similar to keystone species that interact with a large
number of other bacteria and hence have a significant influence on the commu-
nity’s structure and function [90]. Testing the effects of biotic variables on
microorganisms that cannot be grown on their own will be particularly difficult.
For example, strong interactions can ‘link’ organisms to other microbes (e.g.
mycoparasitism). Network analysis is particularly useful for identifying related
microorganisms and ‘hub’ microorganisms, which are comparable to keystone
species in that they interact with a large number of other bacteria and so have a
considerable impact on the structure and function of the community [90].
1.4 Core Plant Microbiome and Metagenome
Targeted investigations of agricultural microbiomes in the field, in addition to

model systems and culture collections, provide critical fundamental information
that can lead to innovation in a variety of ways. First, identifying the ‘core’ microbiome –
the set of microbial species identified in the majority of samples of a given set of
plants [91–95] – will aid in identifying plant-associated microorganisms that
should be prioritized for future research, inclusion in culture collections, and
manipulation studies. Although the plant microbiota is varied, not all of these
microorganisms play functionally essential roles in the biology of their hosts.
Researchers can filter out transitory connections and focus on stable taxa that have
a better chance of impacting host phenotype by defining the core microbiome. In
contrast to very deep sequencing of a few plant microbiomes, culture-independent
surveys (such as sequencing internal transcribed spacer (ITS) and 16S rRNA ampli-
cons) of large numbers of microbiomes of the same plant species from a variety of
environments would improve progress towards this goal and could be followed-up
by selective culturing of candidate core microbiota. Second, finding a functional
core microbiome in addition to a taxonomically defined core microbiome based on
phylogenetic differences is critical since component bacteria are likely to be adapt-
able. Using metagenomic and metatranscriptomic techniques to uncover shared
projected functions that are likely relevant for the collection of plants investigated,
the functional core may be found [96]. Extrapolating functional information from
phylogenetic marker genes like ITS offers only a limited amount of information
while metatranscriptomics, metaproteomics, and metabolomics, on the other
hand, show the functional community phenotype, whereas focused metagenomics
of functional genes and shotgun metagenomics provide a deeper understanding of
community functional potential [97]. We can detect fundamental community
functionality and the level of taxonomic functional redundancy using a com-
bination of multi-omic techniques across large sample sizes of plants. Third,
comparing core microbiomes or metagenomes between important plant groups

and genotypes of the same plant species may reveal host-driven variations in
microbiome assembly. The soil is the most important source of microorganisms
that make up the plant microbiome [93], while there have been rare reports of
seed-borne vertical transmission [98], and the atmosphere also contributes to
above-ground plant microbiota [99]. Plants of various species or genotypes gener-
ate a mainly shared core microbiota, derived from the same environmental ‘inocu-
lum’ at taxonomic levels of family and above. To what extent does the host impact
microbiome assembly, and is within-species host genetic variation sufficient for
breeding superior microbiome associations? Broad-sense heritability is often found
to be between 5 and 7% [7, 100], implying that the potential of microbiome-related
variables to react to conventional artificial selection may be restricted. Microbiomes
might, however, be altered to boost heredity [101], and focused breeding efforts
might be successful if we understand the host molecular basis of microbiome con-
struction. Fourth, comparing the core microbiota of genetically diverse plant
groupings may uncover plant genes and functional features that drive microbiome
formation. Although plant functional features such as cuticle composition [102],
root length and exudates [103, 104], and plant defences (immunity) [105] have
been implicated, the methods by which hosts winnow the ambient community to
establish their microbiota are not entirely known. Individual microorganisms (e.g.
rhizobia, mycorrhizae) may have indirect impacts on later-arriving microorgan-
isms, whereas cuticle and root characteristics should directly impact colonization
by a wide variety of microbial species [106]. Furthermore, crop species or cultivars
that consistently assemble various microbiomes may be relying on their microbiota
to meet a variety of demands, especially if the plants are suited to distinct environ-
mental difficulties [107].
1.5 Interactions Among the Microbes, Environment,

and Management
All organisms are inhabited by microorganisms including archaea, bacteria,

fungi, and viruses; this microbiota plays a key role in host health and develop-
ment [104, 108]. The microbiome associated with plants is considered its second
genome. It is determinant for plant health, growth, fitness, and consequently pro-
ductivity [109]. Each environment associated with the plant – rhizosphere, endo-
sphere, and phyllosphere – presents a specific microbial community with specific
functions [4].
These culture-independent methods show that plant microbiome can reach
densities greater than the number of plant cells and also greater expressed genes
than the host cells. Metagenomics analysis using next-generation sequencing
1.5 Interactions Among the Microbes, Environment, and Managemen 15
technologies shows that only 5% of bacteria have been cultured by current methods,
revealing how many microorganisms and their functions remain unknown [110].
The first step in plant–microbe interaction is microbial recognition of plant exu-
dates in the soil. There is a hypothesis that plants are able to recruit microorgan-
ism by plant exudates, which are composed of amino acids, carbohydrates, and
organic acids that can vary according to the plant and its biotic or abiotic condi-
tions [111]. Different plants select specific microbial communities as reported by
Berg et al. [104] when comparing rhizosphere colonization of two medicinal
plants: chamomile (Matricaria chamomilla) and nightshade (Solanum distichum);
despite being cultivated under similar conditions, they presented different struc-
tural (analysing 16S rRNA genes) and functional (analysing nitrogen-fixing nifH
genes) microbial communities. Moreover, plant exudate of the same plant varies
according to plant developmental stages selecting specific microbial communi-
ties [112]. Researchers already identified some plant exudate compounds respon-
sible for specific interactions such as flavonoids in Legume-Rhizobia [48] and
Strigolactone as a signal molecule for AMF [113].
Reinhold-Hurek et al. [114] proposed a model for microorganism colonization.
In bulk soil, the microbial community presents a great diversity and is influenced
only by soil type and environmental factors. Getting closer to plant roots (rhizos-
phere), where there are root exudates, there are fewer species and a more special-
ized community. And only a few species are able to enter plant root and establish
in the plant. Furthermore, after entering the plant, microbial community varies
among the different organs: top leaves, fruits, bottom leaves, flowers, stems, and
roots [115]. Mutualistic microorganisms can protect plants from pathogen either
by inducing plant resistance or by antibiosis. The ISR in plants leads to high toler-
ance to pathogens. There are soils that even if there is the pathogen the disease
does not occur; the mechanisms of these disease suppressives are still being inves-
tigated. In this way, Mendes et al. [116] analysed the microbiome of a soil suppres-
sive to the fungal pathogen Rhizoctonia solani that causes damping off in several
agricultural crops. Using a 16S rDNA oligonucleotide microarray (PhyloChip),
they were able to identify more than 33 000 bacterial and archaeal taxa in the
sugar beet seedlings rhizosphere grown in suppressive soil and in conductive soil.
These analyses revealed the bacterial groups present only in the suppressive soil.
The authors reported that γ-proteobacteria, especially Pseudomonadaceae, were
all more abundant in suppressive soil than in conductive soil, focusing thereby on
this bacterial group. Using random transposon mutagenesis technic in
Pseudomonas sp. they were able to identify genes responsible for the biosynthesis
of an antifungal: nine amino acid chlorinated lipopeptide produced by
Pseudomonas sp. and that controls the pathogen. From the same PhyloChip diver-
sity analysis, Cordovez et al. [117] identified other antifungal, this time produced
by rhizosphere-associated streptomycetes. These Streptomyces isolates were able
to produce chemically diverse volatile organic compounds (VOCs) with an antifungal

activity as well as plant growth-promoting properties. Showing that different
bacteria groups can have similar roles in the same environment, another example
was reported by Ardanov et al. [118] who showed that the inoculation of
Methylobacterium strains also protected plants against pathogen attack and
affected endophyte communities. Therefore, using this concept, researchers
started inoculating plants with a pool of microorganism with complementary
traits, for example with different mechanisms of control; however, it is a challenge
to find the right players to be inoculated [119].
In order to define which microorganisms should be inoculated, several
approaches were used. The first approach seeks to define a core microbiome of
a healthy host, or understand the function of microbiomes by sequencing
approach, that can be followed by experiments on gnotobiotic host manipulat-
ing the microbiome with a selection factor (for example, antibiotics, salinity,
and UV light) or transferring microbiomes between hosts [101]. In this way,
researchers are starting to study ‘microbiome engineering’, modulating micro-
bial community. This modulation can occur either by performing plant breed-
ing programmes selecting a beneficial interaction between plant lines and
rhizosphere microbiome or by redirecting rhizosphere microbiome by stimu-
lating or introducing beneficial microorganisms [101, 119]. The microbiome
engineering can occur by altering ecological processes such as modulation in
community diversity and structure changing microbe–interaction networks
and by altering the evolutionary processes that include extinction of microbial
species in the microbiome, horizontal gene transfer, and mutations that can
restructure microbial genomes [120].
Summarizing, plant phenotype is the sum of plant response to the environment
and to the present microbiome (including endophytes and pathogens); this micro-
biome also responds to the environment and interacts with each other [121].
Mendes and Raaijmakers [108] suggest a similarity between gut and plant rhizos-
phere microbiomes. They are both open systems, with a gradient of oxygen, water,
and pH resulting in a large number and diversity of microorganism due to the
different existing conditions. There are differences between gut and plant rhizos-
phere microbiome composition, therefore there are some similarities related to
nutrient acquisition, immune system modulation, and protection against infec-
tions. Berg et al. [122] point seven similarities between host-associated microbi-
ome ecology, among them: different abiotic conditions shape the structure of
microbial communities; host and its microbiome co-evolute; core microbiome can
be transmitted vertically; during life cycle, the microbiome structure varies; host-
associated microbiomes are composed of bacteria, archaea, and eukaryotic micro-
organisms; functional diversity is key in a microbiome; and microbial diversity is
lost by human interventions.
1.5.1 Secondary Metabolism

Microorganisms produce a large variety of compounds known as secondary
metabolites that do not play an essential role in growth, development, and repro-
duction of the producing organism. Nevertheless, these metabolites are often bio-
active compounds and can perform important functions in defence, competition,
signalling, and ecological interactions [123, 124]. To establish a microbial interac-
tion network, microorganisms usually respond by metabolic exchange, which
leads to complex regulatory responses involving the biosynthesis of secondary
metabolites. These interactions can be parasitic, antagonistic, or competitive and
the metabolites involved and their functions have been specially studied recently
as a result of the advent of tools such as metabolomics and imaging mass spec-
trometry (IMS) technology [125, 126]. Siderophores are related to competitive and
cooperative microbial interactions and can also play other roles, such as signalling
and antibiotic activity [127, 128]. Hopanoids play an important role in bacterial
interaction, conferring tolerance and improving the adaptation of bacteria in dif-
ferent environments [129, 130]. In fungi, the compounds differentially regulated
in an interaction are often bioactive secondary metabolites, such as diketopipera-
zines, trichothecenes, atranones, and polyketides [131, 132]. Nevertheless, there is
still a lot to understand about the mechanisms involved and the role of many
secondary metabolites and genes differentially expressed during the interaction.
In this section, we present examples of studies on secondary metabolites involved
in different types of microbial interactions.
1.5.2 Endophyte–Phytopathogen–Plant Interaction

The metabolites and mechanisms involved in the interactions between endo-
phyte, phytopathogen, and host plant are still very unclear and are predicted to
involve many secondary metabolites. Endophytic fungi are known to produce a
large variety of bioactive secondary metabolites [133, 134] that are probably
related to the endophyte complex interactions with the host and the phytopatho-
gens and can perform important ecological functions, for example, in the plant
development (as growth promoters) and in defence, acting against phytopatho-
gens [135, 136]. This interaction has been studied in co-cultures of the phy-
topathogen Moniliophthora roreri and the endophyte Trichoderma harzianum
that cohabit in cacao plants [127]. T. harzianum is extensively used as a biocon-
trol agent and has known ability to antagonize M. roreri. Tata et al. [127] identi-
fied four secondary metabolites (T39 butenolide, harzianolide, sorbicillinol, and
an unknown substance) whose production was dependent on the phytopathogen
presence and was spatially localized in the interaction zone [127]. T39 butenolide
and harzianolide have been reported to have antifungal activity. Sorbicillinol is
an intermediate in the biosynthesis of bisorbicillinoids, a family of secondary

metabolites that present diverse activities [137].
Trichoderma atroviride, commonly used as a biocontrol agent, produces acetic
acid-related indole compounds that may stimulate plant growth. Colonization of
Arabidopsis roots by T. atroviride promotes growth and enhances systemic disease
resistance conferring resistance against hemibiotrophic and necrotrophic phy-
topathogens [138]. Other co-cultured studies were performed with bacteria.
Araújo et al. [139] isolated a great number of Methylobacterium strains from
asymptomatic citrus plants (with Xylella fastidiosa but without disease); then
Lacava et al. [140] showed that Methylobacterium mesophilicum SR1.6/6 and
Curtobacterium sp. ER1.6/6 isolated from health and asymptomatic plants inhib-
ited the growth of the phytopathogen X. fastidiosa, the causal agent of citrus var-
iegated chlorosis. Moreover, transcriptional profile of X. fastidiosa was evaluated
during in vitro co-cultivation with a citrus endophytic strain of M. mesophilicum.
It was shown that genes related to growth, such as genes involved in DNA replica-
tion and protein synthesis, were downregulated, while genes related to energy
production, stress, transport, and motility, such as fumarate hydratase, dihy-
drolipoamide dehydrogenase (Krebs cycle), pilY transporter, clpP peptidase, acri-
flavin resistance, and toluene tolerance genes, were upregulated [141].
Another approach to study endophyte–phytopathogen plant interaction is
based on the genome sequencing and transposon mutagenesis of an endophyte
strain of Burkholderia seminalis, which suppresses orchid leaf necrosis by
Burkholderia gladioli, which revealed eight loci related to biological control. A
web cluster related to the synthesis of extracellular polysaccharides of the bacte-
rial capsule was identified [142]. Extracellular polysaccharides are known to be
key factors in bacterial–host interactions [143, 144]. In addition, gene clusters
putatively related to indole-acetic acid and ET biosynthesis were identified in the
sequenced genome of the endophyte strain, suggesting that this strain might
interact with the plant by altering hormone metabolism [142].
1.5.3 Hopanoid
Hopanoids compose the cell membrane of some bacteria, [145] presenting the same
function of eukaryotes cholesterol. They are responsible for stabilization of the mem-
brane and regulate its fluidity and permeability [146]. Experiments that knockout bio-
synthesis genes such as hnpF (squalene hopene cyclase: shc) gene show that the
absence of hopanoids does not influence bacterial growth [146, 147] but affects toler-
ance to several stress conditions, such as extremely acidic environments [148] or toxic
compounds such as dichloromethane (DCM) [149]; it also affects the resistance to
antibiotics [64] and antimicrobial lipopeptide [63], playing a role in multidrug trans-
port [83] and bacterial motility [84]. Hopanoids act in increasing bacteria tolerance to
adverse environments, conferring resistance to stress conditions including extreme

pH and temperature and exposure to detergents and antibiotics [129, 130]. In this way,
hopanoids may be involved in bacteria–plant interaction, being responsible for adap-
tation of bacteria in aerobic microenvironment and low pH culture medium [131] as
well as involved in nitrogen metabolism in Frankia sp. [150]. For example, a type of
hopanoids produced by the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens is
essential for its symbiosis with the host Aeschynomene afraspera, a tropical legume. In
this case, the synthesis of C35 hopanoids is related to evasion of plant defence, utiliza-
tion of host photosynthates, and nitrogen fixation [151].
1.5.4 Parasitic Interaction

The study of the mycoparasitic interaction between Stachybotrys elegans and
R. solani revealed many secondary metabolites differentially expressed in the
interaction [132]. During the interaction, S. elegans produces cell-wall-degrading
enzymes and expresses genes associated with parasitism [152, 153] while R. solani
responds with an elevated level of the pyridoxal reductase-encoding gene [152].
A metabolomic study showed the profile of the induced secondary metabolites
during the interaction. It showed a significant effect of the mycoparasite on
R. solani metabolism: the biosynthesis of many antimicrobial compounds were
downregulated, possibly as a result of the interaction, and only a few diketopip-
erazines were induced [132]. Diketopiperazines are known to have antimicrobial
properties, among others biological activities [154]. The mycoparasite S. elegans
produced several mycotoxins, mainly trichothecenes and atranones. It was hypoth-
esized that the trichothecenes were triggered by R. solani and were responsible for
the alteration in its metabolism, growth, and development [132]. Trichothecenes
are a major class of mycotoxins and have been reported to inhibit eukaryotic pro-
tein biosynthesis and generate oxidative stress.
1.5.5 Microbial Community’s Interaction

Actinomycetes are noteworthy as producers of many natural products (NPs) with
a wide range of bioactivities [126]. A study on Streptomyces coelicolor interacting
with other actinomycetes showed that most of the compounds produced in each
interaction was unique, revealing a differential response in each case. Many
unknown molecules and an extended family of acyl-desferrioxamine siderophores
never described before in S. coelicolor were identified. They identified 227 com-
pounds differentially produced in interactions; half of these were known metabo-
lites: prodiginines, actinorhodins, coelichelins, and acyl-desferrioxamines. Thus,
actinomycetes interspecies interaction seems to be very specific and complex [155].
It has been shown that fungal–bacterial interactions can lead to the production of
specific fungal secondary metabolites and not only diffusible compounds act in
this communication but also there is a contribution from physical interaction [156].
Schroeckh et al. [157] demonstrated that an intimate physical interaction between
Aspergillus nidulans and the actinomycete Streptomyces rapamycinicus leads to the
activation of fungal secondary metabolite genes related to the production of
aromatic polyketides, which were otherwise silent. A PKS gene required for the
biosynthesis of the archetypal polyketide orsellinic acid, lecanoric acid (typical
lichen metabolite), and the compounds F-9775A and F-9775B (cathepsin K inhibitors)
was identified [156]. It was later reported that alterations in fungal histone acety-
lation via the Saga/Ada complex are triggered by the actinomycete leading to the
induction of the otherwise silent PKS cluster. This result shows that bacteria can
trigger alterations of histone acetylation in fungi [156].
1.5.6 Siderophore
The production and acquisition of siderophores by microorganisms is a crucial
mechanism to obtain iron. Many microorganisms secrete siderophores in the
environment that when loaded are recognized by cell surface receptors and then
transported into the microbial cell [158]. Thus, they are related to competitive and
cooperative microbial interactions. In addition, many siderophores can also pre-
sent other functions, for example, they can function as sequesters of a variety of
metals and even heavy metal toxins, as signalling molecules, as agents in regulat-
ing oxidative stress, and as antibiotics, which were reviewed by Johnstone and
Nolan. In some Pseudomonas species, a group of siderophores called pyoverdines
is essential for infection and biofilm formation, probably helping to regulate bac-
terial growth [159]. Pyoverdines have been reported to act as signalling molecules
triggering a cascade that results in the production of several virulence factors,
such as exotoxin A, PrpL endoprotease, and pyoverdine itself [160].
In the marine environment, exogenous siderophores affect the synthesis of
induced siderophores and other iron acquisition mechanisms by other microbial
species, working as signalling compounds that influence the growth of marine
bacteria under iron-limited conditions. Many strains of marine bacteria were
reported to produce siderophores and iron-regulated outer membrane proteins
only in the presence of exogenous siderophores produced by other species, such
as N,N-bis(2,3-dihydroxybenzoyl)-O-serylserine from a Vibrio sp., even under very
low iron concentrations [161].
1.5.7 Symbiotic Interaction

A remarkably complex inter-kingdom interaction is the symbiotic relationship
between Burkholderia, a genus of bacteria, and Rhizopus, a genus of phytopathogen
1.6 Microbiome Innovation in Agriculture: Insect Pest Managemen 21
fungi that causes rice seedling blight. The endosymbiotic bacteria Burkholderia spp.
is responsible for the production of the phytotoxin rhizoxin, the causal agent of rice
seedling blight [162]. It was reported that in the absence of the endosymbiont,
Rhizopus is not capable of producing spores, indicating that the fungus is dependent
on factors produced by the symbiont to complete its life cycle [163]. This complex
symbiont–pathogen–plant interaction is still poorly understood regarding the metab-
olites and mechanisms involved in the communication and interaction. A study on
exopolysaccharide (EPS), which usually plays key roles in interactions, produced by
Burkholderia rhizoxinica described a previously unknown structure of EPS. However,
the loss of EPS production did not affect the endosymbiotic interaction with Rhizopus
microsporus, as shown by a targeted knockout mutant experiment [164]. B. gladioli
produces enacyloxins (polyketides with potent antibiotic activity) in co-culture with
R. microsporus. The fungus induces the growth of B. gladioli resulting in an increased
production of bongkrekic acid, which inhibited the growth of the fungus [164].
1.6 Microbiome Innovation in Agriculture: Insect

Pest Management
Insects are associated with diverse microbial communities and in many cases,
these associations are crucial for insect survival and development. Symbiotic
microbes in the gut, hemolymph, as well as in specialized cells carry an arsenal of
enzymes that provide specialized services to the insect hosts [165]. Supplies of
essential nutrients (particularly amino acids and B vitamins) by endosymbionts
have been well documented in a number of crop pests, particularly plant sap-
sucking Hemipteran insects such as aphids, whiteflies, and psyllids [166–169],
and in human disease vectors and urban pests such as tsetse flies in the genus
Glossina and the common bed bug (Cimex lectularius). Some symbionts can
degrade complex polysaccharides or recycle nitrogen for insects, such as the ter-
mites [170, 171] and cockroaches [172, 173]. The production of antimicrobials by
symbionts aids the immune system to fight against invading pathogens, as was
shown in the beewolf digger wasps [174] and cotton leafworm [175]. Besides
nutritional and immune services, symbionts can shape the ecological interactions
between insects and their natural enemies. For instance, the secondary symbiont
of aphids Hamiltonella defensa increased the chance of host survival from parasi-
toid wasp attacks by disrupting wasp embryogenesis, mediated by its bacteriophage-
encoded toxins [176–180]. H. defensa was also shown to attenuate volatile release
in aphid-infested plants, thus reducing parasitic wasp recruitment [181]. Similarly,
symbiont manipulation of plant physiology that facilitates insect colonization was
observed in whiteflies and the Colorado potato beetle [182, 183]. Modification of
body colour by facultative symbionts may determine aphid susceptibility to
predation or parasitism [184]. In particular, Rickettsiella infection in the pea aphid

Acyrthosiphon pisum increased the synthesis of blue-green polycyclic quinone
pigments, turning the host from red to green. This symbiotic-dependent colour
variation is believed to affect the aphid’s relative risks between predation and par-
asitism, as their predators such as the ladybird beetles preferentially prey on the
red morphs, while parasitoids preferentially attack the green morphs [185–187].
Termites [171, 172] and cockroaches have symbionts that can digest complex pol-
ysaccharides or recycle nitrogen for them [173]. As proven in the beewolf digger
wasps [174] and cotton leafworms [174], symbionts produce antimicrobials that
help the immune system combat invading infections [175]. In different insect
pests, symbionts were also shown to impact pesticide resistance. Resistance to
organophosphorous pesticides is related to the direct detoxification by their sym-
bionts in the beans bug (Riptortus pedestric) and the eastern fruit fly (Bactrocera
dorsalis) [176, 177]. Bacillus thuringiensis (Bt) insecticide action was demon-
strated to depend on the presence of symbiotic mid-gout bacteria in gypsy moth
larvae Lymantria dispar [188]. Field microbiome studies showed that Bt resist-
ance in bollworm cotton (Helicoverpa armigera) was related with different micro-
biome compositions [178–180]. The diamond back moth (Plutella xylostella) was
diagnosed with varying levels of sensitivity to Chlorpyrifos treated with antibiot-
ics and then decolonized with various gut-associated bacteria [189, 190]. In mos-
quitoes that demonstrated decreased lethality of pesticides, various intestinal
commensal bacteria were found to have been contributing to pesticide resist-
ance [191]. Collective data suggest that bacteria have a greater role as previously
assumed in the formation of insect behaviour [192]. Long-term scatter, egg posi-
tion, mattress, hosted search, and kin recognition are insect behaviours that are
found to be altered by microorganisms [193]. Studies further imply that microbi-
omes might alter host behaviour by the formation of host neuro-endocrine circuit-
acted metabolites [194–196], a phenomenon termed the ‘gut-brain axis’. There has
been a lot of study on the gut-brain axis, with the majority of it being on mam-
malian systems. This field, however, is still in its infancy. A recent study found
that the microbiome of the Drosophila melanogaster alters the host’s olfactory-
guided foraging preferences towards meals with various microbial content [197].
Farine et al. [198] and Qiao et al. [199] showed similar microbiome-priming
effects on fly behaviour in following investigations. Scientists have achieved two
breakthroughs in insect microbiome research thanks to advances in high-
throughput sequencing and functional genomics: (i) investigate previously uni-
dentified microbiomes in a wider range of insects, resulting in a better knowledge
of the host and environmental variables that influence insect microbiome diver-
sity and composition – some examples include microbial communities associated
with Drosophilid and Tephritid fruit flies [200–202], ants [203, 204], bees [205],
mosquitoes [206, 207], ticks [208], beetles [209], and midges [210], among others;
and (ii) assign particular microbial taxa or consortia with microbiome roles. While
insect microbiomes differ in terms of diversity and stability, there is widespread
agreement that microbial effect on insect invasive characteristics exists. For exam-
ple, the invasiveness of the sweet potato whitefly (Bemisia tabaci) was aided by the
introduction of a Rickettsia sp. into the pest population (from 1% infected in 2000
to 97% in 2006), which resulted in faster development, a higher survival rate to
adulthood, and increased host fecundity [197, 211–214]; it was found that the
microbiome of the strong lab model D. melanogaster accelerates larval develop-
ment, impacts host foraging choice, and reproduction [197, 213]. Drosophila
suzukii, often known as spotted wing drosophila (SWD), is a tiny fruit pest that
relies on the microbiome to flourish [215]. Microbial symbiosis was initially dis-
covered in the olive fruit fly Bactrocera oleae, a very damaging agricultural pest
belonging to the Tephritidae family [216]. Unlike other fruit-feeding Bactrocera
species, B. oleae has an obligatory bacterial symbiont (Candidatus Erwinia daci-
cola) that is maintained in the midgut caeca of the larvae. The symbiont is yet to
be grown, but studies have demonstrated that it aids fly growth and reproduction
by delivering necessary amino acids and metabolizing urea from a variety of
sources, including bird droppings, making nitrogen accessible to adult flies [217,
218]. It also aids the development of larvae in unripe olives by inhibiting the pro-
duction of oleuropein, a plant defence molecule [217, 219]. The symbiont was
shown to be missing in domesticated B. oleae grown on antibiotic-laced artificial
medium, illustrating the influence of upbringing on symbiont selection [220].
Pantoea sp. and Burkholderia sp. are two more bacterial species found in the intes-
tines of B. oleae, although their nutritional significance is unknown [202]. The
medfly (Ceratitis capitata) and apple maggot fly both have microbiome-dependent
larval development (Rhagoletis pomonella). Diazotrophs that express the nitrogen
reductase gene (nifH) in the stomach of medflies are involved in microbial food
supply [221]. Citrobacter, Klebsiella, Pectobacteria, Enterobacter, and Pantoea are
among the bacteria found in medflies [200]. When provided as probiotics, the
community has been found to boost fly growth, reproduction, and lifespan, as
well as boost male copulatory success [200, 218]. A modest but persistent popula-
tion associated with the medfly stomach contains Pseudomonas spp., in addition
to the dominating Enterobacteriaceae. Malacrin et al. [222] used a metabarcoding
strategy to find changes in the microbial community at different instar stages of
the medfly [222]. Burkholderia was discovered to be prevalent in early instars and
adults, and it has been postulated that it may play a role in nitrogen fixation in
Tetraponera ants [223]. Similarly, bacteria from the genera Sphingomonas
and Pseudomonas, as well as an unidentified bacterium from the family
Methylobacteraceae, were found to be more abundant in late instars of the med-
fly, whereas bacteria from the families Leuconostoc, Weissella, Acetobacter,
Gluconobacter, and an unidentified bacterium from the family Xanthomonadaceae,
were more abundant at pupal stage. In addition, medflies fed on different host
plants had a diverse microbial community. Malacrin et al. [222] observed that
medfly larvae fed Ficus carica fruits had Acinetobacter and Gluconobacter, whereas
Acetobacter and Leuconostoc were more prevalent when given Prunus persica
(peaches). It has been proposed that Acinetobacter and Gluconostoc are involved in
phenolic glycoside detoxification [224]. Similarly, enterobacteria such as Pantoea,
Klebsiella, and Enterobacter are found in the guts of apple maggot flies [225].
During oviposition, microorganisms are deposited into the fruit, supplying neces-
sary nutrients and proteins for larval growth [225, 226]. Symbiotic bacteria
enhance larval development in several Tephritids of the subfamilies Dacinae and
Trypetinae by metabolizing carbohydrates, raising organic nitrogen levels, and
producing vitamins [217, 225]. However, their functions in adult flies are
unclear [218].
1.6.1 Manipulation of Insect-Associated Microbiomes

for Pest Management
The contribution of microbiomes to a wide range of insect invasiveness-related
features implies a wealth of resources that might be targeted for pest control. The
use of biochemicals to eradicate or disrupt insect symbiosis is a straightforward
method [227]. Antibiotics such as tetracycline and penicillin, for example, have
been demonstrated to render tsetse flies infertile by inhibiting the obligatory
mutualist Wigglesworthia, obstruct the development of young ticks, and reduce
adult tick reproduction by lowering their symbiont load [228]. Antimicrobial pep-
tides (AMPs) have also been investigated for manipulating insect symbionts,
albeit they are more typically utilized to combat human or plant infections trans-
mitted by insects [229]. Insect innate immunity relies heavily on AMPs (which
include a variety of amphiphilic and cationic oligopeptides). They provide immu-
nity against bacteria, fungi, and viruses, among other microorganisms. Cecropin
was the first AMP recovered from Hylaophora cecropia pupae [230, 231], and
since then over 150 AMPs have been purified from insects [232]; for example, the
α-helical peptides (e.g. moricin and cecropin), cysteine-rich peptides (e.g. defen-
sin and drosomycin), proline-rich peptides (e.g. apidaecin, drosocin, and lebocin),
and glycine-rich proteins (e.g. apidaecin, drosocin [e.g. attacin and gloverin).
Some AMPs (such as defensins, cecropins, attacins, and proline-rich peptides) are
found in all insect orders, whereas others (such as moricin and gloverin) are
exclusively found in particular insect orders (e.g. Lepidoptera). AMPs’ antibacte-
rial activity is due to their positively charged surface, which allows them to bind
negatively charged microbial surfaces via charge–charge interactions, disrupting
bacterial cell wall integrity [233]. Resistance to bacterial, fungal, and certain
eukaryotic parasites has been conferred by AMPs in plants and insects. Insect
defensins (gallerimycin from Galleria mellonella) and cecropin (sarcotoxin-IA

from Sarcophaga peregrine), for example, have been found to provide harmful
fungal resistance in transgenic tobacco [234]. In rice and tomato plants, trans-
genic production of cecropins has also been demonstrated to give resistance to
fungal and bacterial diseases [235, 236]. Cecropins have also been shown to be
effective against protozoan parasites including Plasmodium and Trypanosoma [237,
238]. The use of transgenic cecropin expression in Anopheles gambiae, a human
parasite vector, has been found to lower the quantity of Plasmodium berghei
oocysts by 60% [239]. Furthermore, transgenic co-expression of defensin-A and
cecropin-A in Aedes aegypti has been found to prevent Plasmodium parasite trans-
mission cooperatively [240]. Non-target effects, which can lead to disturbance of
the natural microbiome of non-target insects, are a significant drawback of utiliz-
ing antibiotics or AMPs. Low bioavailability, instability, and antimicrobial resist-
ance are some of the other drawbacks of employing AMPs [241]. Nonetheless,
approaches such as fusion with antigen-specific antibody fragments [242], protein
engineering, and synthetic biology approaches can be used to create AMPs with
improved specificities (e.g. substitution of amino acids, chemical modifica-
tions) [243]. Nanotechnology-assisted distribution of AMPs to many biological
systems is also being investigated [244]. Insect trait manipulation can also be
accomplished by introducing a foreign microbe or replacing a symbiont with
another bacterium. In stink bugs and aphids, experimental substitution of certain
cultivated and uncultured insect symbionts has been demonstrated. According to
Hosokawa et al. [245], exchanging the Ishikawaella symbionts between the stink
insect Megacopta punctatissima, a common pest of soybean and other legumes,
and a closely related non-pest species, Megacopta cribraria, resulted in poor
M. punctatissima egg hatching on the plants. The major symbiont Buchnera was
replaced with a new genotype by microinjection in the pea aphid in one experi-
ment, and the pest’s heat tolerance was altered [246].
1.6.2 Incompatible Insect Technique (IIT)

Several techniques have been developed to produce gnotobiotic insects in the lab,
including Drosophila [213], mosquitoes [247], and honey bees [248]. Cleaning
insect eggs to remove maternally deposited bacteria on the surface or treating lar-
val or adult insects with antibiotics, followed by feeding on food seeded with cul-
tured microbes or microbe-laden materials were all common parts of the technique
(e.g. faecal transplantation). While the gnotobiotic approach has aided in the dis-
covery of microbiome impact on insect traits such as development, physiology,
behaviour, and insecticide resistance [190, 213, 247–249], its application in pest
management is still largely theoretical. The use of Wolbachia to control mosqui-
toes and mosquito-borne disease pathogens is one notable exception. Wolbachia is
a widespread, vertically transmitted endosymbiont found in arthropods, infecting

nearly 60% of all insects [250]. The bacteria are most recognized for inducing cyto-
plasmic incompatibility (CI), feminization, parthenogenesis, and male death in
its hosts [251]. Wolbachia-infected females have a reproductive advantage over
uninfected females because their sex ratios are skewed towards females, making it
easier for them to spread across a community. Wolbachia-induced CI is used in the
incompatible insect technique (IIT) to reduce mosquitoes and other insect pests
[259[251, 252]. CI causes embryonic death [253] and can be caused unidirection-
ally in crossings between Wolbachia-infected males and uninfected females, or
bidirectionally in crosses between infected animals carrying different Wolbachia
strains [254]. As demonstrated in A. aegypti, Wolbachia-induced sterility has little
effect on male mating competitiveness or survival [255]. Wolbachia-infected
males are returned into the wild on a regular basis at IIT [256, 257]. Several insect
pests, including Rhagoletis cerasi, C. capitata, the tsetse fly, and disease vectors,
such as Culex pipiens, Aedes albopictus, and Culex quinquefasciatus, have been
studied extensively using IIT [256, 257]. Wolbachia can be transinfected into a
new host that is not natively infected with CI-inducing Wolbachia strains [258–260].
Wolbachia strain wSuz, as naturally, infects D. suzukii but does not cause
CI [261–263]. D. suzukii has been successfully introduced to two CI-inducing
Wolbachia strains (wHa and wTei) from different Drosophila species, paving the
way for IIT for this pest [261, 264]. By distorting sex ratios toward females,
Wolbachia-infected females have a reproductive advantage over uninfected
females, facilitating their propagation in a population. The incompatible insect
technique (IIT) employs Wolbachia-induced CI as a strategy to control mosqui-
toes and other insect pests [251, 252]. In addition, because host genotype influ-
ences Wolbachia density and phenotypic manifestation of infection in hosts,
including CI, the genotype of IIT insects should be examined [265, 266]. Recent
research has made substantial progress in identifying the molecular mechanisms
that cause Wolbachia-mediated CI. When expressed dually in uninfected males,
the wMel genes cifA and cifB (encoded by WO prophage) functionally repeated
CI, according to LePage et al. [267]. Both genes are unable to cause CI on their
own. Further research revealed that transgenic production of the cifA gene in
Drosophila recovers CI and eliminates the embryonic lethality induced by wMel
Wolbachia [268]. Beckmann et al. [269] found that the Wolbachia deubiquitylat-
ing enzymes (DUB) cidA and cidB combine to cause CI in transgenic Drosophila.
Aside with CI, Wolbachia’s pathogen-blocking capabilities are another key feature
for mosquito control. When transinfected with Wolbachia generated from
Drosophila or other mosquitoes including A. albopictus and C. quinquefasciatus,
A. aegypti, the vector for several clinically important arboviruses, showed dramat-
ically decreased competence for dengue, chikungunya, yellow fever, and Zika
viruses [270–273], as well as Plasmodium and filarial nematodes [274, 275].
The specific mechanism of Wolbachia-mediated pathogen inhibition is still being

researched. Priming of the immune system, alterations in cholesterol and lipid
droplet formation and trafficking [276], and (viral) RNA degradation are among
the possibilities that have been offered [277]. In the year 2011, in the wild in
Cairns, Australia, A. aegypti harbouring the wMel strain were released, marking
the first experiment of microbiome modification of a wild insect population with
the objective of lowering vector competence [278]. The Wolbachia infection has
firmly established in the mosquito population, according to a follow-up experi-
ment two years later [278]. More crucially, the discharge effectively halted dengue
transmission in Cairns and its environs in northern Queensland, Australia.
Meanwhile, Wolbachia frequencies in the original Cairns populations are at 95%,
with a 96% decline in dengue incidence as of late 2019. Wolbachia has now been
found in northern Queensland, as well as in Yogyakarta, Indonesia, and Kuala
Lumpur, Malaysia, as a result of further releases [279–281].
1.6.3 Paratransgenesis
Paratransgenesis, a similar method that has gained popularity in recent years,
involves genetically modifying microorganisms to express desired effects in
insects [282–284]. Paratransgenesis avoids the problems of fitness cost associated
with introducing a transgene into insects and transgenic instability in insect
genomes by not changing the insects (i.e. transgenesis). This method is best for
bacteria that can be cultivated, altered, and reintroduced into insect hosts with
ease. Although paratransgenesis was first postulated in the early 1990s, the major-
ity of study has focused on human disease vectors and a few Hemipteran crop
pests. Beard et al. [285, 286] demonstrated that the triatomine bug’s gut symbiont
Rhodococcus rhodnii may be genetically engineered to express effector molecules
(cecropin A and similar pore-forming molecules) against the protozoan
Trypanosoma cruzi, which causes Chagas disease. Inoculating eggshells or food
with excrement seeded with the designed symbiont allows the symbiont to be
introduced to insect offspring. Durvasula et al. [287] used an anti-trypanosome
single-chain antibody to alter the symbiont and found a considerable decrease in
parasite burden. Following the positive findings of laboratory investigations, field
trials were conducted to assess the transmission efficiency of modified R. rhodnii
to the triatomine bug using CRUZIGARD, a simulated triatomine-faecal sub-
stance made of an inert guar gum matrix painted with India ink [288]. To manage
Rhodnius prolixus, a study recently combined paratransgenesis with RNA inter-
ference (RNAi) technology. In R. prolixus, oral administration of an Escherichia
coli strain HT115 or R. rhodnii engineered to express dsRNA targeting the antioxi-
dant genes-heme-binding protein (RHBP) and catalase (CAT) genes caused sys-
temic RNAi to silence these genes, resulting in poor nymph development and
reduced female fecundity [289]. Using the engineered symbiont Sodalis glossinidius,
which produced antigen-binding molecules targeting Trypanosoma brucei,
the causative agent of sleeping sickness, similar paratransgenic techniques have
been explored on tsetse flies. Sodalis may be transferred vertically by the milk
glands and is present in the hemolymph, midgut, and milk gland [290, 291]. Using
bacteria and fungi obtained from mosquito midguts and ovaries, different para-
transgenic techniques have been investigated in mosquitoes to limit the transfer
of malaria-causing Plasmodium parasites. The Gram-negative Asaia bogorensis
was chosen for paratransgenesis against P. berghei because it has been demon-
strated to survive in mosquito midguts and spread swiftly both vertically and hori-
zontally within a population [292, 293]. The siderophore receptor gene was fused
with anti-plasmodial effector genes to create genetically engineered Asaia strains.
The scorpine AMP and a synthetic antiPbs21 scFv-Shiva1 immunotoxin made up
of a single-chain antibody (scFv) against the P. berghei ookinete surface protein
21-Shiva1 fusion protein were among the genes identified. Anopheles stephensi
mosquitoes fed with the altered Asaia and challenged with P. berghei-infected
blood showed a substantial reduction in parasite growth [294]. Pantoea agglomer-
ans, common mosquito symbiotic bacteria, was previously modified to produce
anti-Plasmodium effector proteins utilizing an E. coli-derived Type I hemolysin
secretion system. In the midgut of Anopheles mosquitoes, these modified P. agglo-
merans strains were shown to suppresses the growth of P. falciparum and
P. berghei [295]. Metarhizium anisopliae, an entomopathogenic fungus, has also
been engineered to release the antibiotic scorpine and anti-plasmodial peptide
SM1, which inhibits Plasmodium parasite growth [196]. Novel Serratia sp. AS1
colonizes the ovaries and gut of A. stephensi as AS1 strain was both sexually and
vertically transmitted, persisting for at least three generations. Mosquitoes
infected with an engineered Serratia AS1 containing five different anti-
plasmodium effector molecules (Shiva1, a cecropin-like synthetic AMP; MP2,
midgut peptide 2; EPIP, enolase–plasminogen interaction peptide (lysine-rich
enolase peptide); scorpine, scorpion Pandinus imperator venom AMP; and
mPLA2, inactive bee venom phospholipase A2) displayed a reduction in the
oocyte load by 93% [296]. The Glassy-winged sharpshooter (GWSS), Homalodisca
coagulata, demonstrates the potential of paratransgenesis in crop protection
against insect pests or insect-vectored illnesses. GWSS is a vector for X. fastidiosa,
a bacterial pathogen that causes Pierce’s disease in grapes by manufacturing EPSs,
which assist the infection to colonize the xylem of its host plant and restrict the
flow of the xylem [297–299]. Alcaligenes xylosoxidans var. denitrificans (Axd) was
identified as a candidate for genetic modification among the several bacterial spe-
cies identified from GWSS. It has always been detected in the xylem of host plants,
in the same spot where the pathogen lives. GWSS was successfully given with
genetically engineered Axd harbouring a DsRed fluorescent protein gene from
injected stems. It was discovered to invade the foregut of insects, implying that a
paratransgenic method to eradicate X. fastidiosa from GWSS is possible [298].
However, as this genus of bacteria has been recognized as a nosocomial human
pathogen linked in causing lung infection in cystic fibrosis patients, the usage of
altered Axd in plants has significant downsides [300]. To alleviate the safety con-
cern, an endophytic bacteria from grapes, P. agglomerans E325 (an EPA-approved
agent for managing fire blight in pears and apples), was genetically engineered to
express anti-Xylella effector proteins melittin and a scorpine-like AMP, and colo-
nized the foregut of GWSS using an artificial feeding system (AFS) [301]. In addi-
tion, in simulated field settings, targeted administration of recombinant
P. agglomerans E325 to the gut of GWSS utilizing a microencapsulation approach
was established to treat Pierce’s illness. The microencapsulation approach might
be effective in the field since it might restrict the transmission of alien genetic
material [301]. Leonard et al. [302] devised a paratransgenic technique in honey-
bees and showed in the lab that it increased bee survival against viral infection
and Varroa mites. The researchers created symbiotic gut bacteria called
Snodgrassella alvi that produces dsRNA that targets bee, virus, and mite genes.
The selection of microorganisms, the genetic design, and the implementation of
the treated insects all have a role in the success of gnotobiotic or paratransgenic
techniques for insect pest management. Another barrier is the association’s stabil-
ity, which should be particular to target insects or innocuous to non-target hosts.
Although persistent association guarantees that microbial-mediated effects on
host insects are long-lasting, certain microorganisms may be ‘lost’ from the insects
owing to environmental selection pressure or antagonistic interactions with other
germs. Despite these cautions, it is expected that gnotobiotic and paratransgenic
insect research and development will continue to expand.
1.6.4 Exploiting the Chemical Inventories of Microbiomes

to Develop New Biopesticides
The evolution of pesticide resistance is a major barrier for crop security, since many
insect pests have no other treatment options. The difficulties of turning a lead com-
pound into a product that can pass tight environmental and safety laws have made
the development of new synthetic pesticides more expensive and complicated.
According to a recent research, developing a synthetic pesticide in the United States
currently costs over $300 million and takes about 12 years [200]. The demand for
novel insecticidal chemicals is necessitated by the need to address resistance issues
and improve sustainable agriculture. The effort to produce innovative insecticides
with little environmental effect has sparked renewed interest in biopesticides in
recent years (i.e. pesticides based on living organisms or their NPs, including their
genes and metabolites). The global market for biopesticides is now estimated at
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‘I was surprised at this sudden rencontre, Marie. I know not why,
but I did not expect that we should ever meet again. It certainly was
not my wish, although you will not give me credit for the cause.’
‘And what is that?’
‘I will tell you. You know I left Paris for Liége, my native place,
some time ago. I have since then followed my profession there, and
am about to be married. My intended lives at Mezières, whence I am
now returning from a visit.’
‘And you ought to forget me,’ replied Marie: ‘it is right to do so.’
Then she added, ‘Do you remember the last evening we met,
Camille?’
‘It would be difficult to forget it. I have the scar here on my arm
from Monsieur de Sainte-Croix’s sword. Where is he—at Paris still?’
‘I know not,’ answered the Marchioness, with a violent effort to
conceal her emotion; ‘it is long since we have met.’
‘He may be alive or dead, for aught I could say to the contrary,’
said Theria. ‘I never hear from Paris now.’
‘He knows nothing then,’ thought the Marchioness.
‘But how is it I find you here?’ continued Theria; ‘so far from home,
and alone?’
‘Alas! Camille, it is a sad story, and some day you shall know
everything. I have been compelled to fly from Paris—from my
creditors—to avoid a prison. The separation from my husband and
children drove me to seek any excitement that would drown my
wretchedness. I played deeply, and I am ruined.’
‘Are you pursued?’
‘I believe the authorities are close upon my track. I only left Paris
the evening before last. Your old friend Philippe Glazer came with
me to Offemont, and from that place I have travelled alone.’
‘I think you might have chosen a better resting-place,’ said Theria.
‘This is the principal hotel, and the first to which the police would
come. I shall wait here until my horse is rested, and then push on to-
night, if possible, to Dinant; for I must be at Liége to-morrow. Will you
accompany me?’
‘Again upon the road!’ murmured his companion in accents of
despair. ‘My strength has nearly deserted me!’
‘It will be safer for you, if things are as you state,’ replied Camille.
‘You will have passed the frontier, and be three leagues nearer the
termination of your journey. We will sup together if you please, Marie,
and talk it over; I shall not start for an hour yet. Mass! how the wind
is shrieking along the market-place!’
‘I will go with you,’ said Marie, after a little deliberation. ‘I could not
bear to be left here now, wretched and utterly deserted as I am. The
sight of you has recalled so many old feelings, that——’
‘Understand me, Marie,’ interrupted Camille, ‘the past must be
never again alluded to between us. I have told you my position, and
if we meet, it can only be as friends.’
‘It shall be as you wish, Camille,’ replied the Marchioness with a
sigh. ‘I will not give you cause for the lightest rebuke.’
Some of the people of the inn appeared at that moment, and at
Camille’s orders laid out a table for supper. When they left the room
he said—
‘Have you no other dress? In my quiet vehicle your rich costume
would at least excite curiosity; and the more unobserved we are the
safer.’
‘I have provided against any suspicion,’ said Marie; and taking up
the bundle she had brought with her, she left the room, returning
within five minutes attired as a paysanne of the Forêt de l’Aigue. Her
hair, which she usually wore in showering ringlets about her neck
and shoulders, was knotted and disordered by her journey, and she
stood before a large mirror in the room, to put it up beneath a small
country cap, first letting fall its entire flowing length, with a coquetry
that was intended to produce its effect upon Theria. But Camille’s
affections were fixed at present rather on a brioche that adorned the
table, and the effect was lost.
Whilst thus occupied, an unusual stir was heard in the street below
the inn. Marie, alive to every sound, again rushed to the window, and
to her dismay perceived that her worst fears were realised. A
mounted escort of guards had surrounded a carriage, in which, by
the lights they carried, she could plainly recognise Desgrais, and two
other exempts. He had closely followed her, making up for the time
lost in the wild-goose chase towards Beauvais by double speed as
soon as he found himself on the right track; and, as Camille had
imagined, came first to the principal hotel.
‘I am lost!’ she exclaimed, as she retreated from the window. ‘They
have traced me!’
‘Not yet,’ said Camille, jumping up. ‘But you must be off directly.
Where is your passport?’
A cry of terror broke from Marie’s lips at the question. She had left
home without one, forgetting that it would be demanded at the
frontier.
‘Never mind,’ cried Theria; ‘this way. We can get into the court
before they enter by this staircase, and thence to some of the back
streets. You must run every risk, if you wish to escape; though I
should imagine, for a matter of debt, they would not be very hard
upon you. Come—come!’
Little persuasion was needed to induce Marie to accompany her
new guide. They flew down the small flight of stairs indicated by
Theria, and were quickly in the street in the rear of the hotel, whence
a few turns conducted them to the river side, where the Meuse was
chafing amidst the huge blocks of ice which had floated down its
stream, and were gathering into one solid mass.
‘If you could but cross the river,’ he said, ‘we should be safe. But a
boat could not make its way amidst the ice. We will try it, however, if
you choose.’
‘I am ready,’ said Marie. ‘The chance is a desperate one either
way.’
‘We must not be particular about what craft we take,’ said Theria,
‘so long as it remains undiscovered. Here is one I think will do.’
A small boat had been hauled on to the bank, which Theria directly
launched through the brittle ice close to the shore; and then,
assisting Marie to enter it, he got in himself, and pushed off with one
of the stretchers. So rapidly had everything taken place, that before
the Marchioness well understood what they were about, she found
herself with Theria half across the river.
It was not very dark. One or two lights were gleaming and
struggling with the wind along the edge of the river; and the frosty
brightness of the stars was sufficient to enable them to discern
surrounding objects. The huge blocks of ice kept floating about them,
at times turning their boat completely round, and at last a
conglomeration of these masses hemmed them in, threatening
entirely to arrest their farther progress. Theria made a few strenuous
efforts to set the boat free, but in vain. Another and another block
joined the body, until the entire mass, wedging itself in with some
fixed groups that extended a third of the way across the river,
became altogether immovable.
‘Pheuh!’ said Theria, as, after a few laborious attempts to get the
boat out, he threw down his piece of board, and saw the futility of his
work. ‘What can we do now? We are fairly trapped.’
‘It is all over!’ exclaimed Marie, as she gazed at the gloomy
masses, about which the cold feathery spray of the river was
dashing, terrible to look at in the obscurity. ‘We shall be kept here
until daylight, and then be captured.’
‘If we are, I shall be mistaken,’ said Theria. ‘The ice ought to make
a bridge, although a slippery one.’
He tried to gain a footing upon one or two of the blocks; but they
turned round as he touched them. At last he found one larger and
firmer than the rest—a conglomerate of several pieces, forming a
perfect iceberg—and this was frozen to some others that had been
arrested in their progress by one or two piles just under water. It was
extremely hazardous; but their only chance was to endeavour to
reach the bank by this treacherous passage. Theria stepped
carefully from the boat on to the block, which, somewhat depressed
in the middle, offered a safer platform to stand upon than those of a
more irregular shape. Then, assured of its stability, he gave his hand
to the Marchioness, and bidding her to trust herself entirely to his
guidance, assisted her on to the ice, moving with extreme caution,
and sideways towards the bank. The least slip of the foot or
overbalance of weight would at once have been fatal to both; but,
fortunately, the severity of the frost had so bound the masses to
each other, that in little more than a minute their perilous journey was
accomplished, and they stood on the firm land on the other side of
the river. The cold had kept all within doors, so that they were not
observed by any passers by, and the darkness hid them from the
view of the sentinels on the adjacent fortifications.
Camille directly led Marie to a small cabaret on the quay, and told
her to await his return, whilst he went back to the hotel by the bridge
—having his passport en règle, and being, moreover, slightly known
to the authorities. His absence had scarcely been noticed at the ‘Ane
Doré’ in the confusion, although they were eagerly seeking the
Marchioness; so he ordered out his horse and little conveyance, and
drove over the bridge to the spot where he had left Marie. Here she
joined him, and they then set off together to Dinant, the first town in
Belgium on crossing the frontier, where they arrived in two hours.
Now Marie determined at all hazards to stop. She had meant to do
so at Givet, had it been practicable, for her strength would hold out
no longer; indeed, for the last ten miles of her journey, she had been
in a complete state of stupefaction from want of rest, after the trials
she had undergone. Theria went to another house to avoid any
suspicion, recommending her to post onward in the morning, so as
to reach Liége before Desgrais could get any order for her
‘extradition’ from the Conseil des Soixante in that city. The chances
were in favour of her security; for no one had seen her leave Givet,
nor would the passport books afford any information as to her route.
Meantime Desgrais had learned sufficient at the ‘Ane Doré’ to
convince him that the Marchioness had been there; and the
discovery of the garments she had left at the hotel at once decided
him. But she had again slipped through his hands, and this time
without leaving a trace of her journey behind her. He immediately
sent his archers round to the commissaries of police and the
barriers; but no passport had been seen that night, nor were the
guards aware that any one had crossed the bridge since dark,
except Theria, whom they mentioned. But he knew that the
Marchioness had the passage of the frontier for her object, and that
Liége, as the nearest place of importance, would in all probability be
the end of her journey; and consequently, leaving a portion of his
men at Givet, with orders to make the strictest investigation at all the
hotels and small inns in the neighbourhood, he went on the same
night to Dinant, actually sleeping in that town within two hundred
yards of his object.
Marie was up as soon as there was daylight enough to proceed on
her journey. Twenty leagues were now all that remained between her
and Liége, and these she meant to traverse before night. The rest of
some hours had refreshed her, bodily and mentally, and she was
once more ready to encounter any difficulties her further progress
might bring forth. The exempt never heard of the departure (which
he immediately knew to be that of the Marchioness), until three or
four hours after she had left Dinant; and then, still at a loss to
account for the manner in which she had contrived to elude the
police authorities at Givet, he ordered out a carriage and horses, and
started after her with all the speed his money and authority could
command, leaving his archers behind—with the exception of two
who accompanied him—with orders to follow him as hastily as their
means would permit.
Empanne, Havelange, Nandrin—all were passed without any
circumstance occurring to obstruct Marie’s flight; and the gloom of
the winter’s night was closing fast about her as the carriage came
within the last mile of Liége. It was here, as she looked behind her
through the small window at the back of the vehicle, to see if there
were any signs of pursuit on the road—which had been her sole
occupation during the day—that she first perceived two gleaming
lights in the distance, evidently following her. She urged on the
postilions, and a turn of the road hid them from her view. Then they
were again visible, and apparently nearer; directly the brow of the
hill, as she descended once more, shut them out, and the next
minute she saw them gaining upon her during every interval of
perfect darkness. Swiftly as she was flying along the road, it was
evident that the other party was more than a match for her attelage
in speed; and perceiving from this that every effort was being made
to come up with her, she concluded that it was Desgrais.
Lashed and goaded to madness, her horses flew on like the wind,
as from the front of the carriage she promised an additional reward
every instant to their riders if they brought her to Liége before the
other traveller. But Desgrais—for it was he—was equally on the alert.
On the first intimation that a carriage was in sight on the road before
them, he had left the interior, and, clinging to the front of the voiture,
was urging his own people on as earnestly as the Marchioness, until
the uproar of cries and cracking whips was plainly audible to the
terrified inmate of the first vehicle. Tearing uphill, until the breathless
horses almost fell from being overtasked—anon racing down, with a
precipitancy that threatened annihilation every instant—and then
flying along the level road, so close together, that the steam from the
animals in the carriage of the Marchioness was still visible in the
gleam of the lamps belonging to Desgrais—did the chase continue.
At last they entered Liége, and the pursuit now became doubly
exciting from the cries of the postilions as they traversed the
glooming streets at a fearful pace, cracking their whips as they
whirled them above their heads, and shouting in an unearthly
manner to warn the passengers of their advent. A charette in the
road offered a temporary check to Marie’s carriage, and Desgrais the
next instant was close up to her. But nearer he could not come; for
the width of the thoroughfare would not allow the two vehicles to go
abreast. They were, however, coming to a broader street, and then
Marie knew he would pass her. To avoid this, and gain a minute of
time—for every second now was worth the price of her life—she
collected some straw from the interior of her coach, and tied it into a
bundle with her handkerchief; then lighting it at the lamp of the
carriage, she leaned out of the window, and threw it, blazing, directly
in front of the leaders of the other voiture. The horse on which the
postilion was riding reared up in fright, and directly threw him; his
fellow backed as well, and the wheelers coming over them, they
were all thrown together in a terrible confusion before the carriage,
which by its own impetus came partly on them. In an instant
Desgrais leaped upon his feet—for the shock had also thrown him
upon the ground—and clearing the rider from the stirrups, he cut the
traces with his poniard, and getting the horse upon his legs, vaulted
into the saddle, leaving the rest of his equipage to the care of the
archers who were inside. The carriage of the Marchioness was not
fifty yards ahead, as it turned towards the convent she had indicated
to the drivers. Once more everything depended on a few seconds,
and Desgrais goaded the poor animal with the point of his weapon to
spur it onwards, as the horses of his intended prisoner, equally
urged, kept tearing on towards the goal. At last they stopped at the
door of the convent, and as its heavy bell sounded with a loud and
violent peal, the exempt came up to the carriage.
He sprang from his horse, and tore down, rather than opened, the
door nearest the road, and seized the Marchioness by her mantle. At
that instant the gate of the convent opened, as she jumped from the
carriage and entered the lodge, leaving the garment in the hand of
the exempt. Desgrais rushed through the vehicle, and was about to
follow her, when she seized a cross from the porch, and held it
towards him with a smile of triumph, that threw an expression of
demonaic beauty over her features.
‘You dare not touch me!’ she cried; ‘or you are lost, body and soul!’
With an oath, Desgrais fell back before the sacred emblem. Marie
had thrown herself upon the Church, and claimed a sanctuary. An
impassable barrier was between them, and the whole of his toil to
arrest her had gone for nothing. The chance had been lost, in a
pursuit of nearly one hundred leagues, by half a minute.
CHAPTER XXXIII.
THE END OF LACHAUSSÉE
Whilst all this turmoil had been going on, Paris was no less a scene
of excitement; indeed, it was greater, inasmuch as it affected a larger
number of persons. The awful death of Sainte-Croix, and the
discoveries which had arisen from the unexpected revelation of the
casket, furnished sufficient matter for conversation to all the gossips
of the good city. Maître Glazer’s shop was more than ever besieged
by the curious bourgeoisie, as he was supposed to be better
acquainted than any one else, not even excepting the commissary of
police, with the circumstances of the event. But it was remarked that
Philippe preserved a perfect silence respecting the share which the
Marchioness of Brinvilliers was known to have had in the
transactions of the newly-discovered poisoners. He always avoided
the most distant allusion to the catastrophe, and even when Maître
Picard wished to push his questions very closely—half in his
capacity of public functionary, half as a private gossip—the young
student generally cut all his queries so very short, that Picard almost
imagined he must have been one of the parties implicated.
‘For, look you, messieurs,’ the little chapelier would say, when he
got out of Philippe’s ear-shot, and was traversing the Place Maubert,
‘Madame de Brinvilliers had as many accomplices as our good King
Louis—whom Montespan preserve!—has sweethearts. Else, whence
came the powerful armed force which unhorsed me on the road to
Le Bourget?’
‘She had dealings with the sorcerers,’ observed a neighbour.
‘I believe it,’ replied M. Picard. ‘I heard of her with Exili, who is
about to suffer at the gibbet of Montfaucon, the night M. de Sainte-
Croix died. And the exempt’s guards, who returned to Paris, have
affirmed that she flew past them on a whirlwind whilst they halted at
Le Bourget. She will never be taken—no: the devil would save her
from the centre of the Chambre Ardente itself, even if M. La Reynie
had the care of her. Allons! buvons! it is a wicked world!’
And then the little bourgeois and his neighbours turned into the
nearest tavern, and, whatever might be the time of day at their
entrance, never appeared until after curfew had sounded, when
Maître Picard was usually conducted home to the Rue de la Harpe
by the Gascon, Jean Blacquart, whose unwillingness to engage in
personal encounter was scarcely sufficient to keep the chapelier
from pot-valiantly embroiling himself with everybody unarmed that he
chanced to meet. Our business is not, however, so much with these
personages just at present; but with those of whom we have not
heard for some little time.
Night was closing round the gloomy precincts of the Cimetière des
Innocents—mysterious, cold, cheerless. The snow lay upon the
burial-ground, and clung to the decaying wreaths and garlands that
rotted on the iron crosses which started up from the earth. The
solemn and dreary place was doubly desolate in the wintry trance of
nature. In the centre of the cemetery a tall obelisk arose, and on the
summit of this, some fifteen feet from the ground, was a large
lantern, from which a pale light gleamed over the abodes of the
dead, throwing its rays sufficiently far to reveal a ghastly procession
of corpses, of all ages and professions, painted on the walls and
covered charnels in which the wealthier classes were interred who
chose to carry their exclusiveness into the very grave. This danse
macabre, or dance of death, was then rapidly becoming invisible at
different stages of its march. At various parts of the enclosure small
lamps struggled with the wind, as they hung before images of the
Virgin placed in niches of the walls and tombs, and lights were
visible in the higher windows of the crowded, and not unpicturesque,
buildings that enclosed the cemetery; but elsewhere everything was
dark, and the place was untenanted but by the dead.
One figure, however, might have been seen kneeling at a fresh
grave for some time, in spite of the inclemency of the weather. And
about this the snow had been cleared away; the chaplets on the
small cross were fresh, and a few dark evergreens were planted at
the head and foot. A scroll in the ironwork bore the inscription, ‘Cy
giste Gaudin de Sainte-Croix, qui trépassa, la vingt-neuvième année
de son âge.’ It was the tomb of the guilty lover of the Marchioness of
Brinvilliers, and the solitary mourner was Louise Gauthier.
Of all with whom Sainte-Croix had been on terms of intimacy, not
one had cared to make inquiry after him, when the report of his
death was first promulgated, but the Languedocian. But Louise,
assisted by Benoit (with whom she had returned to live, since the
evening at the Hôtel de Cluny, when she again fell in with him), had
seen the body taken from the dismal vault below the Palais des
Thermes to his old abode in the Rue des Bernardins. She had been
the solitary mourner when his body was rudely consigned to that part
of the ground allotted to those for whom no consecrated rites were
offered; and her own hands afterwards had adorned the grave—the
only one thus distinguished in this division of the cemetery—with the
humble tributes that were about it. All this she had done without one
tear or expression of the wretchedness that was breaking her heart;
but when it was accomplished, she gave full vent to her pent-up
feelings, and was accustomed to seek the cemetery every evening,
weeping and praying in the terrible solitude of the burial-place, over
the grave whose narrow limits comprised her world.
It was past the time of curfew; but the city of Paris had not the air
of quietude which it usually bore at this period of the night. The
murmur of a distant multitude could be heard mingling with the
occasional solemn tolling of some hoarse and deep-mouthed bell,
and now and then the roll of drums calling troops together. Louise
had been some hours in the cemetery, when she was surprised by
the appearance of Benoit and his wife, who had come to seek her,
alarmed at her unusual stay from home, although they were aware of
the locality in which she was most likely to be found. The honest
couple had started off together to bring her back; and now, assisting
her to rise, had persuaded her to return with them.
As they got into the Rue des Lombards, on their way towards the
river, a sudden rush of people in great numbers separated them from
one another, and they were obliged to fall in with the stream, which,
increasing at every corner of a fresh thoroughfare, almost carried
them off their legs. Louise addressed a few questions to some that
she came in contact with, but no answer was returned; all appeared
too anxious to hurry onward. Soon the crowd became more dense in
the narrow streets, and the confusion and jostling was increased by
the mounted guard who pressed on through the people, almost
riding them down, amidst the screams of the women and curses of
the men, who only received a few blows in return. She was now
entirely borne onward by the multitude, and in the dense mass of
people could scarcely look up to see in what direction she was being
impelled, until she found herself close to the Grand Châtelet.
The whole of the carrefour was lined with troops carrying cressets,
so that it was light as day; and in the centre a scaffold was erected,
on which one or two figures were standing. One of these was a
priest, the others were masked, and held what appeared in the
distance to be long staves in their hands. Louise’s heart sickened as
she foresaw that she was about to be present at an execution, and
one of the most terrible kind. There was no headsman’s block on the
platform; but some apparatus could be seen upon the floor, but a few
inches in height. A wretch was about to be broken on the wheel.
Suddenly the murmurs of the people ceased; lights moved in slow
procession from the Châtelet, and the voices of monks could be
heard chaunting a requiem. They advanced between lines of troops
towards the scaffold, and then the criminal could be distinctly seen.
He was not walking, however, between them, nor was he dragged on
a sledge, but borne on a species of bier, raised on the shoulders of
some of the soldiery; from which the spectators knew that the
question had been undergone, and the rack had left its victim
crippled, with dislocated limbs. By the men in masks he was lifted on
to the platform, and then a yell from the vast multitude assembled
broke the silence that had just reigned. It was a terrible cry of ferocity
and denunciation.
Louise could scarcely speak; but she asked a female who was
close to her the name of the criminal.
‘One of the poisoners,’ replied the woman; ‘his name is
Lachaussée. He will make up for Sainte-Croix’s cheating us out of
his execution. And the Marchioness of Brinvilliers will follow, when
she is caught. Oh! these are brave times! I should like to have seen
Sainte-Croix broken. They say he was handsome; and that he would
have held out to the last. Hist!’
The noise of the multitude ceased as the priest advanced to the
edge of the scaffold and addressed them. His words could only be
heard by the few around him; but they were carried from one to the
other, and were to the effect that the criminal had refused to confess,
after having undergone the question both ordinary and extraordinary;
that his own guilt had been sufficiently proved; but that none of his
accomplices had been named, except his master and instructor,
Monsieur Gaudin de Sainte-Croix, upon whom a just retribution had
fallen. The last judgment of the law would now be carried into effect,
but the coup de grace would be withheld until the criminal had
confessed all that he was known to be acquainted with respecting
his presumed accomplice, the Marchioness of Brinvilliers, now in
sanctuary, as it was supposed, at a convent beyond the frontier.
There was an awful silence. The wretched man was seized by the
other figures on the scaffold and placed upon the wheel, and the
next minute the staff in the hands of one of the executioners was
raised. It descended with a dull, heavy sound, distinctly audible at
every part of the square, as was the sharp cry of agony that burst
from the lips of the culprit. The priest stooped down, and appeared to
commune with him; but in a few seconds he rose again, and the
blow was repeated, followed by the same scream, but less piercing
than before. Another and another followed, and then a conversation
of greater length took place between the criminal and his confessor.
The monk advanced again to the front of the scaffold, and waving his
hand, stopped the murmur that was rising from the crowd as they
commented on the proceedings.
‘The criminal Lachaussée has confessed,’ he said. ‘He
acknowledges his guilt, and also that of Madame Marie-Marguerite
d’Aubray, Marchioness of Brinvilliers, hitherto suspected, from whom
he owns to have received the poisons with which her two brothers
were murdered. The coup de grace may now be given.’
He held up a crucifix in sight of the writhing object of his speech,
and directed the chief executioner to despatch his victim. The man
again raised the bar, and it descended upon the breast of
Lachaussée, crushing all before it. No cry followed the blow this
time: the death of the wretched man was instantaneous.
The multitude remained silent for a few seconds, as if they were
listening for another cry. But voices were at length heard, first one
and then another, gradually spreading, until the murmur broke forth
into one savage roar of exultation, when they knew that the criminal
had ceased to exist. A clue had been found to the mystery in which
the deaths by poison had long been involved; and now that one of
the participators in the horrible deeds, that had so long baffled the
keenest vigilance of the authorities, had expiated his offence before
their eyes, their satisfaction knew no bounds. And when they had
thus vented their approval of the sight they had just witnessed, they
turned away from the carrefour, and began to leave the spot by the
different outlets.
Louise, who had been scarcely able to sustain herself through the
ghastly scene, was hurried on by the breaking up of the crowd, until
she contrived to get within a porte-cochère, meaning to let them
pass. But she had not been there an instant before she was
recognised by a man in the throng, who had been a servant of
François d’Aubray.
‘Ho!’ cried the fellow, as he saw her by the light of a cresset, ‘here
is another of them. I saw her with Madame de Brinvilliers the night
that her brothers were murdered. She is an empoisonneuse. To
prison with the witch!’
He advanced towards the poor girl as he spoke, whilst the crowd
stopped in their passage. But as he approached her he was seized
by a powerful arm, and, having been twisted round, was flung with
some violence upon the ground.
CHAPTER XXXIV.
THE GAME IS UP—THE TRAP—MARIE RETURNS WITH DESGRAIS TO THE
CONCIERGERIE
Any other officer than Desgrais would have given up further attempts
to arrest the Marchioness, now that she was in the sanctuary of a
convent—in a town, too, where any invasion of the privileges
belonging to a religious house would have been avenged with the
most unrelenting severity. But the exempt felt bitterly the manner in
which he had been more than once duped upon the road, at times
when his prey was completely within his grasp. He was exceedingly
sensitive as regarded his position, and reputation as the most
vigilant officer of the Maréchaussée, and he determined not to enter
Paris again until he could do so accompanied by the Marchioness.
To effect this, he took a lodging in a retired quarter of Liége, and
remained there for a few weeks, dismissing his archers and guards,
with orders to return to Givet, and be in readiness to join him at
Liége upon the shortest notice. To the Marchioness he was
personally unknown. She had not met him above once or twice, and
then without particularly regarding him; and this decided him as to
the course he would pursue. He was young and active; the very
business in which he was constantly engaged had given him
admission into all ranks of society; and he had tact and ready
perception to profit by his observations, and adopt the manners of
any particular class which he found it necessary to assume. He
arranged his plans and, when he imagined sufficient time had
elapsed, proceeded to put them into execution.
To effect the capture he disguised himself in the dress of an abbe,
and presented himself one evening at the gates of the convent in
which Marie had sought shelter, requesting to see her. The porter,
after a slight hesitation, admitted him to the parlour, and in a few
minutes the object of his venture appeared.
The Marchioness had entirely recovered from the fatigues of her
journey. Those who had known her intimately would have remarked
a few lines on her face, resulting from the agitation caused by recent
events; but to others there was still the same girlish, confiding face—
still the same blue lustrous eyes and smooth expansive forehead,
and the rosy lips still half-revealed the same beautiful teeth that had
so dazzled the sight of the gallants, and raised the envy of the
dames of the court at Versailles. She bowed gracefully to Desgrais
as she entered the room, and then in her softest tones inquired ‘to
what chance she was indebted for the honour of a visit from
Monsieur l’Abbé?’
‘I am a poor servant of the Church, madame,’ he replied, ‘and am
returning from a pilgrimage to Rome with relics to be deposited at
the Jacobins, in the Rue St. Honoré. Being detained at Liége upon
matters of ecclesiastical interest, I heard that you were here, and
came to offer my respects.’
‘I have done little to deserve this attention, my holy father,’ said
Marie.
‘You have suffered much undeserved misery, madame,’ answered
Desgrais. ‘You were a supporter of our Church—a good and
charitable lady, as all Paris can vouch; and I should have taken
blame unto myself had I not paid this tribute to your goodness.’
‘Alas! mon père!’ cried Marie; ‘would that the world could think of
me as well as you do. Of what avail has been my past life? You will
find, on your return to Paris, the blackest stories current against me.
A woman, once fallen, has no hope; but every one—those who
would have cringed to her the lowest when she was in her position
being the foremost—will hurry to crush her more utterly, to beat her
lower down. I am lost—for ever!’
‘Yet you should hope that the consciousness of your own
innocence will one day prevail,’ returned the exempt.
‘I have no hope, monsieur. I am alone in this dreary place—alone,
even in the midst of its inmates, as though I were shut out entirely
from the world.’
Desgrais paused for an instant. ‘She has not mentioned her
comrades,’ he said to himself, ‘and she was certainly accompanied
on the road. All accounts agree in this.’
‘You are mistaken, madame,’ he continued aloud. ‘Think. Is there
no one on whom you think you might rely?’
‘What mean you?’ inquired Marie eagerly.
For a few seconds they continued gazing at one another, each
waiting for the other to speak. Desgrais was waiting for some cue,
from which his tact might enable him to proceed, and the
Marchioness was fearful of committing herself by revealing more
than the other knew. Two deep and artful natures were pitted against
each other.
Desgrais was the first to speak. With an assumed expression of
countenance, calculated to impress his companion with the idea that
he understood everything then passing in her mind, and in a voice of
deep meaning, he said—
‘Is there no one, think you, who feels an interest in you? You can
trust me. What communication have you held with the world since
you have been in this retreat?’
‘None, father—on my soul, none.’
‘And have you expected to hear from no one?’ continued Desgrais
in the same tone.
‘Camille!’ exclaimed the Marchioness eagerly. And then, as if
aware she had been indiscreet, she closed her lips forcibly together,
and remained silent.
‘Yes—Camille,’ replied Desgrais, quickly catching at the name.
‘Did you think he had deserted you?’
And he looked cautiously round the parlour, and then placed his
finger on his mouth, as though he was fearful of being overheard.
‘I did not know in what quarter of the town he lived,’ she answered.
‘So,’ thought Desgrais, ‘he is in Liége, then.’
‘And besides,’ she went on, ‘circumstances are changed. He cares
no more for me.’
‘Would you see him?’ asked Desgrais.
The vanity of the woman triumphed over her caution. Camille
Theria, it was evident to Marie, had found his old attachment revive
as they had met again. He had forgotten his fiancée, and was
anxious again to see her.
‘Am I to believe you?’ she asked.
‘You may believe your eyes,’ replied the exempt. ‘He will be at the
tavern of the “Trois Rois” at curfew time to-night.’
‘Why will he not come here?’
‘Would it be advisable? You need fear nothing. I will escort you
from the convent and return with you.’
‘It will compromise your position,’ said Marie.
‘That will be my own affair, madame,’ replied Desgrais. ‘The
weather is unfavourable enough to drive the passengers from the
streets, and the night is dark. No harm can arrive.’
‘What can he want with me?’ said Marie, half speaking to herself,
as she appeared undecided how to act.
‘You will learn all,’ said Desgrais, not trusting himself to speak
further on a subject of which he was so utterly ignorant. ‘But time
presses, and the bells will soon ring out. Come, madame, come.’
Without any other covering than a cloak wrapped about her, and
concealing as much as possible her head and face, Marie yielded to
the persuasions of Desgrais, and, taking his arm, left the convent
unobserved, in the direction of the tavern he had mentioned. The
perfect quietude she had enjoyed since her arrival at the convent
had led her to believe that the French police had entirely given up
their intentions of arresting her. Sainte-Croix, in her fearful
heartlessness, had been already forgotten, and the prospect of a
new conquest—a new victim to her treacherous passions—drew her
on with irresistible attraction.
They traversed the steep and uneven streets of Liége until they
came to the door of the tavern, from whose windows the red
firelights were streaming across the thoroughfare. Desgrais muttered
a few words of excuse for the humble appearance of the place, and
then conducted Marie into the public room.
‘One instant,’ he said. ‘I will ask if he is here.’
He left the room, closing the door behind him, and Marie was a
few moments alone in the apartment. With some slight mistrust, she
listened for his return, and imagined she heard, for a few seconds,
the clank of arms. But this subsided almost immediately, and
Desgrais came back again.
‘Is he not yet here?’ she asked.
‘He is not, madame,’ said Desgrais in an altered tone; ‘nor is it
likely that he will come.’
‘What do you imply?’ exclaimed Marie, somewhat alarmed, and
advancing towards the door.
‘Pardon me, madame,’ said Desgrais, ‘but you cannot pass.’
‘Insolent!’ cried the Marchioness. ‘What does this outrage mean?’
‘That you are my prisoner, madame.’
‘Prisoner! And by whose orders?’
‘By order of his Majesty Louis XIV., King of France,’ cried Desgrais
loudly, as he threw aside his abbe’s robes, and appeared in his
under-clothing as exempt of the guard. ‘Madame, you are mine at
last!’
The words had been the signal to those without, whom he had left
the room to put upon their guard. As he pronounced
‘Then, Madame, you are mine at last!’
them, they rushed into the room, and the Marchioness found herself
surrounded by the archers of the royal guard.
In an instant Marie perceived the trap that had been laid for her.
‘Miscreant!’ she cried, as she rushed at Desgrais in her rage. ‘You
have not yet got your prey within your fangs. I am in a country in
which your authority goes for nought. You cannot arrest me.’
‘Once more, you must pardon me, Madame la Marquise,’ replied
Desgrais, as he drew a paper from his belt. ‘The council of this town
has authorised your extradition, upon a letter from the King. You are
as much our prisoner as though we had arrested you in your own
hôtel in Paris.’
As quick as lightning, upon comprehending the meaning of the
words, Marie drew a poniard from its sheath at the side of one of the
guards, and endeavoured to plunge it into her breast. But her hand
was arrested by another of the party, and the weapon wrested from
her. Baffled in this intention, and in an agony of powerless rage, she
endeavoured to speak, but her mouth refused utterance to the
words, and with a terrible cry she fell senseless upon the ground.
Confiding her to the care of one of his men, and ordering the
others to keep guard without, Desgrais now returned to the convent
in search of further evidence, furnished with proper authority to bring
away whatever he could find. But Marie had little with her. A small
case of letters and papers was, however, discovered under her
pillow, and of this Desgrais immediately took possession. It
contained most important evidence against her—no less than a
confession of the past actions of her life.
In the meantime Marie gradually recovered; but it was some time
before she came completely to herself, from a succession of fainting-
fits supervening one upon another as the least degree of
consciousness returned, and the dreadful reality of her position
broke in upon her. The rough soldier with whom she had been left,
unused to guard such prisoners, and somewhat struck with her
beauty and evidently superior position in life, had been in great
confusion of ideas as to what he ought to do, and had at last called
one of the females attached to the establishment to the aid of the
Marchioness. By some of those trifling remedies which women only
appear to have at command for their own sex, in the like
emergencies, Marie was gradually brought round, and then the
female departed, and she was left alone with her guard—pale and

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Microbiomics and Sustainable Crop

Production 1st Edition Mohammad

Bioinformatics for Everyone 1st Edition Mohammad Yaseen

Handbook of Plant and Crop Physiology 4th Edition

3D Printing and Sustainable Product Development 1st

The Citrus Industry Volume 2 The Production of the Crop

Sustainable Winter Fodder Production Challenges and

Biomanufacturing for Sustainable Production of

Biodiversity Functional Ecosystems and Sustainable Food

Sustainable Production and Applications of Waterborne

Mohammad Yaseen Mir

Limit of Liability/Disclaimer of Warranty

Library of Congress Cataloging-in-Publication Data is applied for

Cover Design: Wiley

Set in 9.5/12.5pt STIXTwoText by Straive, Pondicherry, India

1 Agricultural Microbiomes: Functional and Mechanistic Aspects 1

2 Engineering and Management of Agricultural Microbiomes

3 Approaches and Challenges in Agricultural Microbiome Research 97

4 Perceptive of Rhizosphere Microbiome 111

4.4.3 Metagenomics 131

5 Microbial Communities in Phyllosphere 154

6 Endosphere and Endophyte Communities 193

6.2.7 Aerial Dispersal of the Plant Microbiome 202

7 Core Microbiomes: For Sustainable Agroecosystems 240

7.5.2 Portfolios with Multiple Cores 261

8 Microbiome Mediated: Stress Alleviation in Agroecosystems 272

Microbiomes formed by ages of evolution in soils play an important role in sus-

About the Authors

Dr. Mohammad Yaseen Mir

University of Kashmir for participation in national seminar on ‘Environment

Dr. Saima Hamid

Dr. Saima Hamid holds Doctorate as well as Master’s Degree in Environmental

1.6.7 Combining Microbial-­Based Biopesticides with Nanotechnologies, 36

1.2 ­Model Microbiome–Plant Systems

made progress towards the development of model host–microbiome systems. Many

1.2.1 Plant Perception of Microbes

by yet unexplained mechanisms. Most genes caused by experience of distilled

1.2.2 Molecular Plant

1.2.3 Bacterial Signalling: Quorum Sensing and Symbiosis Factors

1.2.4 Hormone Signalling in Microbe–Host Interactions

responses (abscisic acid [ABA]). Hormone signalling is heavily integrated, and

such quantitative models [49]. Quantitative time-­resolved models are built on

1.2.5 Interactome Network Analysis

interactions was created for membrane proteins [58]. A G-­protein interactome

1.2.6 Transcriptional Regulatory Networks

1.2.7 Metabolic Exchanges and Nutrient Competition in the Soil

1.2.8 Integrated Multi-­omics Modelling

1.2.9 From Systems Biology to Crop Protection

1.3 ­Stability, Resilience, and Assembly of Agricultural

Knowing the identification and functional features of microorganisms present in

to control or regulate the agricultural microbiome. The tremendous diversity of

Network analysis is particularly beneficial for discovering related microorganisms

1.4 ­Core Plant Microbiome and Metagenome

Targeted investigations of agricultural microbiomes in the field, in addition to

comparing core microbiomes or metagenomes between important plant groups

1.5 ­Interactions Among the Microbes, Environment,

All organisms are inhabited by microorganisms including archaea, bacteria,

to produce chemically diverse volatile organic compounds (VOCs) with an antifungal

1.5.1 Secondary Metabolism

1.6.7 Combining Microbial-Based Biopesticides with Nanotechnologies, 36

1.2 Model Microbiome–Plant Systems

such quantitative models [49]. Quantitative time-resolved models are built on

interactions was created for membrane proteins [58]. A G-protein interactome

1.2.8 Integrated Multi-omics Modelling

1.3 Stability, Resilience, and Assembly of Agricultural

1.4 Core Plant Microbiome and Metagenome

1.5 Interactions Among the Microbes, Environment,

1.6 Microbiome Innovation in Agriculture: Insect

1.6.1 Manipulation of Insect-Associated Microbiomes

defensins (gallerimycin from Galleria mellonella) and cecropin (sarcotoxin-IA

The specific mechanism of Wolbachia-mediated pathogen inhibition is still being