New and Future Developments in

Microbial Biotechnology and

Bioengineering: Sustainable
Agriculture: Microorganisms as
Biostimulants Harikesh Bahadur Singh
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NEW AND FUTURE DEVELOPMENTS IN
MICROBIAL BIOTECHNOLOGY AND
BIOENGINEERING
NEW AND FUTURE
DEVELOPMENTS IN
MICROBIAL
BIOTECHNOLOGY
AND
BIOENGINEERING
Sustainable Agriculture:
Microorganisms as Biostimulants
Edited by

Harikesh Bahadur Singh

Department of Biotechnology, GLA University, Mathura, India

Anukool Vaishnav
Department of Biotechnology, GLA University, Mathura, India; Agroecology and Environment,
Agroscope, Zürich, Switzerland
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 Contents

Contributors ix 3. Bacillus rhizobacteria: A versatile

About the Editors xiii
Preface xv
biostimulant for sustainable agriculture
S.R. Prabhukarthikeyan, U. Keerthana, Mathew S Baite,
P. Panneerselvam, Debasis Mitra, R. Naveen Kumar,
1. Role of microorganism as new C. Parameswaran, B. Cayalvizhi, A. Muthu Kumar,
generation plant bio-stimulants: S. Harish, P.C. Rath

An assessment 3.1 Introduction 33

Deepali Shukla, Piyush Shukla, Ashmita Tandon, 3.2 Diversity of Bacillus species 34
Poonam C. Singh, Jayandra Kumar Johri
3.3 Direct mechanism of plant growth
promotion 35
1.1 Background 1
3.4 Indirect mechanism 37
1.2 Introduction of plant bio-stimulants 2
3.5 Future prospects 40
1.3 Basic mechanism of bio-stimulants 2
References 40
1.4 Sources of plant bio-stimulants 2
1.5 Microbes as plant bio-stimulant 3 4. Arbuscular mycorrhizae, a treasured
1.6 Role of microbes in nutrient uptake/
stimulation 8
symbiont to agriculture
1.7 Conclusions 9 Ajay Nair, Archana S. Rao, L. Bhanu, Veena S. More,
K.S. Anantharaju, Sunil S. More
References 10
4.1 Introduction to mycorrhiza 45
2. Exploiting biostimulant properties 4.2 VAM in agriculture 48
of Trichoderma for sustainable 4.3 Application of AMF in bioremediation 55
plant production 4.4 Renaturation and afforestation 56
4.5 Mass production of VAM: the past, present
Ramón Pelagio-Flores, Saraí Esparza-Reynoso, Jesús Salvador
López-Bucio, José López-Bucio and future 57
4.6 Conclusion 59
2.1 Introduction 17 References 59
2.2 Trichoderma metabolism: from decomposers to
plant growth promoters 19 5. Micro and macroalgae: A potential
2.3 Trichoderma-plant chemical dialogue 19 biostimulant for abiotic stress management
2.4 Trichoderma-induced resistance to plant and crop production
pathogens 20 P. Kiruthika Lakshmi, S. Meenakshi
2.5 Trichoderma and plant nutrition 22
2.6 Soil acidification in Trichoderma-plant 5.1 Introduction 63
interactions 25 5.2 Review of literature and recent developments 64
2.7 Salt stress tolerance mediated by 5.3 Conclusion and future prospects 76
Trichoderma 25 References 77
2.8 Conclusions and future prospects 26
References 27

v
vi CONTENTS

6. Fluorescent pseudomonads: 8.5 Genetics involved in nutrient cycling 121

A multifaceted biocontrol agent for 8.6 Conclusions 124
References 126
sustainable agriculture
U. Keerthana, S.R. Prabhukarthikeyan, Mathew S. Baite, 9. Role of soil microbes in
Manoj K. Yadav, R. Naveen Kumar, A. Muthu Kumar, S. Raghu,
S. Aravindan, C.P. Rath
micronutrient solubilization
Bisweswar Gorain, Srijita Paul, Manoj Parihar
6.1 Introduction 88
6.2 Species diversity of Fluorescent 9.1 Introduction 131
Pseudomanads 83 9.2 Importance of micronutrients in plant
6.3 Mechanisms of Fluorescent Pseudomanads 84 nutrition 132
6.4 Future prospects 88 9.3 Sources and pools of micronutrients in soil and
References 89 their significance in plant uptake 133
9.4 Factors affecting the availability of
7. Role of Piriformospora indica in inducing micronutrients 133
9.5 Influence of rhizosphere in micronutrient
soil microbial communities and drought availability 134
stress tolerance in plants 9.6 Soil pH and pE as an indicator of micronutrient
Suryansh Rajput, Preeti Sengupta, Isha Kohli, Ajit Varma, availability 134
Prashant Kumar Singh, Naveen Chandra Joshi 9.7 Micronutrients 135
9.8 Conclusion and future perspectives 145
7.1 Introduction 93
References 146
7.2 Soil microbial communities: benign hidden
players in plant growth 94 10. Sustainable induction of systemic
7.3 P. indica: an overview 95 resistance in response to potential
7.4 Basic mechanisms in plants to counter drought
stress 101
biological control agents in crops
7.5 Morphological and physiological innate Ali Anwar, Efath Shahnaz
responses in plants against drought stress 102
10.1 Introduction 151
7.6 Multidimensional contribution of P. indica in
10.2 Novel scenario of biological control 153
providing tolerance against drought stress 103
10.3 Suppressive soils pathogens 154
7.7 P. indica mediated adaptative responses generated
10.4 Potential in PGPR 155
in rice plants to cope up drought stress 105
10.5 Induction of systemic resistance 157
7.8 Scope of P. indica for the promotion of
10.6 Fungal BCAs 166
sustainable agriculture in xerophytic
10.7 Potental of non-pathogenic strains 167
habitats 106
10.8 Conclusion and future prospects 168
7.9 Conclusion 107
References 169
References 107
11. Psychrophilic microbes: Biodiversity,
8. Microbes-based bio-stimulants towards beneficial role and improvement of
sustainable oilseeds production: Nutrients cold stress in crop plants
recycling and genetics involved Sumit Kumar, Bana Sravani, Tulasi Korra, Lopamudra Behera,
A.L. Singh, Kirti Rani, Rupak Jena, Praveen Kona, Diptanu Datta, Pushpendra Kumar Dhakad, M.K. Yadav
Kiran K. Reddy, K. Gangadhara
11.1 Introduction 177
8.1 Introduction 111 11.2 Historical background 180
8.2 Soil microbes and plant interactions 112 11.3 Biodiversity of psychrophilic microbes 180
8.3 Geochemical changes in plant rhizosphere and 11.4 Mechanisms of adaptation of psychrophilic
release of mineral nutrients 115 microbes 182
8.4 VAM fungi for efficient nutrient acquisition and 11.5 Psychrophilic microbes used in crop
mobilization 119 improvement 186
 CONTENTS
11.6 The beneficial role of psychrophilic microbes in
crop performance 189
11.7 Conclusion and future prospects 192
Reference 193
12. Role of plant-associated bacteria as
bio-stimulants in alleviation of
chromium toxicity in plants
Pratishtha Gupta, Vipin Kumar, Rupa Rani
12.1 Cr toxicity to the environment 199
12.2 Strategies of Cr remediation from contaminated
environment 200
12.3 Plant growth promoting rhizobacteria and their
beneficial traits 200
12.4 Cr induced oxidative stress in plants and
anti-oxidative enzymes 203
12.5 PGPR and phytoremediation 205
12.6 Case study of Cr phytoremediation mediated by
root-associated bacteria 207
12.7 Conclusion 208
References 209
13. Microbe-based plant biostimulants and
their formulations for growth promotion
and stress tolerance in plants
Abhik Mojumdar, Himadri Tanaya Behera,
Smrutiranjan Das, Lopamudra Ray
13.1 Introduction 213
13.2 Microbes as plant biostimulants 215
13.3 Mechanism of development of microbe-based
plant biostimulants 217
13.4 Microbial bioformulation based plant
biostimulants 218
13.5 Microbes as biofertilizers 220
13.6 Biopesticides 222
13.7 Significance of microbes in abiotic and biotic
stress alleviation 223
13.8 Challenges and future prospects 224
13.9 Conclusions 225
References 225
14. Microbial consortia for augmentation
of plant growth–revisiting the promising
approach towards sustainable agriculture
Niharika Ghoghari, Krishna Bharwad, Akshita Champaneria,
Shalini Rajkumar
14.1 Rhizosphere: a nutrient rich niche 234
14.2 Microbial marketing strategies 234
vii
14.3 Plant microbe interactions 234
14.4 Microbe-microbe interactions 236
14.5 Plant probiotics 237
14.6 Plant growth promoting rhizobacteria
(PGPR) 237
14.7 Nitrogen fixation 237
14.8 Mineral acquisition 238
14.9 Phytohormone production 238
14.10 Prevention of diseases and development
of ISR 238
14.11 Biocontrol agents 238
14.12 Biostimulants 239
14.13 Microbial consortia: the dynamics of
co-operation 240
14.14 Binary consortium 243
14.15 Three or multi partner consortium
development 244
14.16 Multi-omics for development of microbial
consortia for plant growth promotion 248
References 250
15. Phosphate solubilization
by microorganisms
Neimar de Freitas Duarte, Christiane Abreu Oliveira Paiva,
Marcela Claudia Pagano, Eduardo Jose Azevedo Correa
15.1 Introduction 257
15.2 Research the selection of phosphate-solubilizing
microbes 268
15.3 Bioinoculants containing strains of P solubilizing
microorganisms and biomaphos - an example of
a successful case in Brazil 269
References 274
16. Fungal endophytes as biostimulants of
secondary metabolism in plants: a
sustainable agricultural practice for
medicinal crops
Tuyelee Das, Abhijit Dey, Devendra Kumar Pandey,
Joginder Singh Panwar, Samapika Nandy
16.1 Introduction 283
16.2 Why do we need to study fungal-medicinal
plant interaction to make secondary
metabolites? 285
16.3 Role of endophytic fungi in production of
secondary metabolites; host-endophyte
relationship 286
16.4 Metabolic interactions of plant
endophytes 299
viii CONTENTS

16.5 Different strategies to exploit fungal endophytes 18.6 Impact of microbiome-based approaches on the

as biostimulants for production of commercially health of plant and soil 338
important plant-derived compounds 301 18.7 Future of microbiome-based approaches in
16.6 Secondary metabolic compounds produced by enhancing soil health: integration of
medicinal plants endophytic fungi in vitro 304 metagenomics and metabolomics approaches
16.7 Conclusion 308 with designing of synthetic communities 339
Acknowledgment 308 18.8 Conclusion 341
References 308 Acknowledgement 342
References 342
17. Plant growth promoting rhizobacteria
from the perspectives of tea 19. Deciphering microbial consortium
plantations and diseases from termite gut for biofertilizer
Stevenson Thabah, S.R. Joshi consortium formulation
Sujit Ghosh, Ambarish S. Vidyarthi, Vijay Kumar, Priyanka Jha
17.1 Introduction 315
17.2 Tea cultivation in India 316 19.1 Introduction 345
17.3 Tea varieties 316 19.2 Material and methods 346
17.4 Shade trees in tea plantations 317 19.3 Results and discussions 347
17.5 Pests and diseases of tea 318 19.4 Conclusion 351
17.6 Tea rhizosphere 319 Acknowledgements 351
17.7 Rhizospheric activity 319 References 351
17.8 Plant growth promoting rhizobacteria
(PGPR) 321 20. Revivification of rhizobacteria-
17.9 PGPR and prospective benefits to tea promoting plant growth for sustainable
plants 322
agricultural development
17.10 PGPR as biocontrol agents in tea
Zahra Dehghanian, Khashayar Habibi, Monire Mones Sardrodi,
cultivation 326
Khosro Balilashaki, Behanm Asgari Lajayer, Tess Astatkie
17.11 Tea plantations and microbial
colonization 326 20.1 Introduction 353
17.12 Conclusion 328 20.2 Rhizosphere soil 354
References 329 20.3 Plant growth promoting rhizobacteria
(PGPR) 354
18. Microbiome-based approaches to 20.4 PGPR in farming 354
enhance soil health in arable land 20.5 Bio-fertilization 355
Shivani Khatri, Shubham Dubey, Shilpi Sharma 20.6 The PGPR biological control agents 355
20.7 Mechanisms of direct 356
18.1 Introduction 333 20.8 Indirect mechanisms 359
18.2 Conventional microbe-based approach for 20.9 Sustainability of agriculture and future
enhancement of soil health 334 perspective 362
18.3 Limitations associated with conventional 20.10 Conclusions 362
approaches 335 References 363
18.4 Microbiome: a brief overview 335
18.5 Approaches used to engineer the Index 369
microbiome 336
 Contributors
K.S. Anantharaju Department of Chemistry, Day-
ananda Sagar Collge of Engineering, Bangalore,
India
Ali Anwar Division of Plant Pathology, Faculty
of Horticulture, SKUAST-Kashmir, Srinagar,
J&K, India
S. Aravindan ICAR-National Rice Research In-
stitute, Cuttack, Odisha, India
Tess Astatkie Faculty of Agriculture, Dalhousie
University, Truro, Canada
Mathew S Baite ICAR-National Rice Research
Institute, Cuttack, Odisha, India
Khosro Balilashaki Department of Horticultur-
al Science, Faculty of Agriculture, University of
Guilan, Guilan, Iran
Himadri Tanaya Behera School of Biotechnol-
ogy, Kalinga Institute of Industrial Technology
(KIIT), Bhubaneswar, Odisha, India
Lopamudra Behera Department of Mycology
and Plant pathology, Institute of Agricultural
Sciences, Banaras Hindu University, Varanasi,
India
L. Bhanu School of Basic and Applied Sciences,
Dayananda Sagar University, Bangalore, India
Krishna Bharwad Institute of Science, Nirma
University, Sarkhej-Gandhinagar Highway,
Ahmedabad Gujarat, India
B. Cayalvizhi ICAR-National Rice Research In-
stitute, Cuttack, Odisha, India
Akshita Champaneria Institute of Science, Nir-
ma University, Sarkhej-Gandhinagar Highway,
Ahmedabad, Gujarat, India
Eduardo Jose Azevedo Correa Agricultural Re-
search Company of Minas Gerais (EPAMIG),
Pitangui, MG
Smrutiranjan Das School of Biotechnology, Ka-
linga Institute of Industrial Technology (KIIT),
Bhubaneswar, Odisha, India
ix
Tuyelee Das Department of Life Sciences, Presi-
dency University, Kolkata, India
Diptanu Datta Department of Plant Pathol-
ogy, Collage of agriculture, Odisha University
of Agriculture and Technology, Bhubaneswar,
Odisha, India
Zahra Dehghanian Department of Biotechnol-
ogy, Faculty of Agriculture, Azarbaijan Shahid
Madani University, Tabriz, Iran
Abhijit Dey Department of Life Sciences, Presi-
dency University, Kolkata, India
Pushpendra Kumar Dhakad Holy Cross Kri-
shi Vigyan Kendra, Canary Hill, Hazaribag,
Jharkhand, India
Neimar de Freitas Duarte Minas Gerais Federal
Institute of Education, Science and Technology
(IFMG), Brazil
Shubham Dubey Department of Biochemical
Engineering and Biotechnology, Indian Insti-
tute of Technology Delhi, Hauz Khas, New
Delhi, India
Saraí Esparza-Reynoso Instituto de Investiga-
ciones Químico-Biológicas, Universidad Mi-
choacana de San Nicolás de Hidalgo, Edificio
B3, Ciudad Universitaria, Morelia, Michoacán,
México
K. Gangadhar ICAR-Directorate of Groundnut
Research, Junagadh, Gujarat, India
Niharika Ghoghari Institute of Science, Nirma
University, Sarkhej-Gandhinagar Highway,
Ahmedabad, Gujarat, India
Sujit Ghosh Department of Botany, J.K. Col-
lege, Purulia, West Bengal, India
Bisweswar Gorain ICAR-Central Soil Salinity
Research Institute RRS Bharuch, Gujarat, India
x CONTRIBUTORS
Pratishtha Gupta Applied Microbiology Labo-
ratory, Department of Environmental Science
and Engineering, Indian Institute of Technol-
ogy (ISM), Dhanbad, Jharkhand, India
Khashayar Habibi Department of Plant Breed-
ing and Biotechnology, Faculty of Agriculture,
University of Tabriz, Tabriz, Iran
S. Harish Tamil Nadu Agricultural University,
Coimbatore, Tamil Nadu, India
Rupak Jena ICAR-Directorate of Groundnut
Research, Junagadh, Gujarat, India
Priyanka Jha Amity Institute of Biotechnology,
Amity University, Major Arterial Road, Action
Area II, Kolkata, West Bengal, India
Jayandra Kumar Johri Department of Plant
Microbe Interaction, CSIR-NBRI, Lucknow, UP,
India
Naveen Chandra Joshi Amity Institute of Mi-
crobial Technology, Amity University, Noida,
Uttar Pradesh, India.
S.R. Joshi Microbiology Laboratory, De-
partment of Biotechnology & Bioinformat-
ics, North-Eastern Hill University, Shillong,
Meghalaya, India
U. Keerthana ICAR-National Rice Research In-
stitute, Cuttack, Odisha, India
Shivani Khatri Department of Biochemical En-
gineering and Biotechnology, Indian Institute
of Technology Delhi, Hauz Khas, New Delhi,
India
Isha Kohli Amity Institute of Microbial Technol-
ogy, Amity University, Noida, Uttar Pradesh,
India
Praveen Kona ICAR-Directorate of Groundnut
Research, Junagadh, Gujarat, India
Tulasi Korra Department of Mycology and
Plant pathology, Institute of Agricultural Sci-
ences, Banaras Hindu University, Varanasi,
India
Sumit Kumar Department of Mycology and
Plant pathology, Institute of Agricultural Sci-
ences, Banaras Hindu University, Varanasi, India
Vipin Kumar Applied Microbiology Laboratory,
Department of Environmental Science and
Engineering, Indian Institute of Technology
(ISM), Dhanbad, Jharkhand, India
Vijay Kumar Department of Biotechnology,
Lovely Faculty of Technology and Sciences,
Lovely Professional University, Phagwara,
Punjab, India
Behanm Asgari Lajayer Health and Environ-
ment Research Center, Tabriz University of
Medical Sciences, Tabriz, Iran
P. Kiruthika Lakshmi Department of Micro-
biology, The Madura College, Madurai, Tamil
Nadu, India
Jesús Salvador López-Bucio CONACYT, Insti-
tuto de Investigaciones Químico-Biológicas,
Universidad Michoacana de San Nicolás de Hi-
dalgo. Edificio B3, Ciudad Universitaria, More-
lia, Michoacán, México
José López-Bucio Instituto de Investigaciones
Químico-Biológicas, Universidad Michoacana
de San Nicolás de Hidalgo, Edificio B3, Ciudad
Universitaria, Morelia, Michoacán, México
S. Meenakshi VMJ School, Madurai, Tamil
Nadu, India
Debasis Mitra ICAR-National Rice Research In-
stitute, Cuttack, Odisha, India
Abhik Mojumdar School of Biotechnology, Ka-
linga Institute of Industrial Technology (KIIT),
Bhubaneswar, Odisha, India
Sunil S. More School of Basic and Applied Sci-
ences, Dayananda Sagar University, Bangalore,
India
Veena S. More Department of Biotechnology,
Sapthagiri College of Engineering, Bangalore,
India
A. Muthu Kumar Faculty of Agriculture, An-
namalai University, AnnamalaiNagar, Tamil
Nadu, India
Ajay Nair School of Basic and Applied Scienc-
es, Dayananda Sagar University, Bangalore,
India
Samapika Nandy Department of Life Sciences,
Presidency University, Kolkata, India
R. Naveen Kumar ICAR-National Rice Re-
search Institute, Cuttack, Odisha, India
 CONTRIBUTORS
Christiane Abreu Oliveira Paiva Brazilian Ag-
ricultural Research Corporation (EMBRAPA),
Sete Lagoas, Minas Gerais, Brazil
Marcela Claudia Pagano Federal University of
Minas Gerais, Belo Horizonte, Minas, Gerais,
Brazil
Devendra Kumar Pandey Department of Bio-
technology, Faculty of Technology and Scienc-
es, Lovely Professional University, Phagwara,
Punjab, India
P. Panneerselvam ICAR-National Rice Research
Institute, Cuttack, Odisha, India
Joginder Singh Panwar Department of Biotech-
nology, Faculty of Technology and Sciences,
Lovely Professional University, Phagwara,
Punjab, India
C. Parameswaran ICAR-National Rice Research
Institute, Cuttack, Odisha, India
Manoj Parihar ICAR-VPKAS, Almora Uttara-
khand, India
Srijita Paul ATC and SAMETI, Ramakrishna
Mission Ashrama, Kolkata, W.B., India
Ramón Pelagio-Flores Facultad de Químico
Farmacobiología, Universidad Michoacana de
San Nicolás de Hidalgo, Morelia, Michoacán,
México
S.R. Prabhukarthikeyan ICAR-National Rice
Research Institute, Cuttack, Odisha, India
S. Raghu ICAR-National Rice Research Insti-
tute, Cuttack, Odisha, India
Shalini Rajkumar Institute of Science, Nirma
University, Sarkhej-Gandhinagar Highway,
Ahmedabad, Gujarat, India
Suryansh Rajput Amity Institute of Microbial
Technology, Amity University, Noida, Uttar
Pradesh, India
Kirti Rani ICAR-Directorate of Groundnut Re-
search, Junagadh, Gujarat, India
Rupa Rani Applied Microbiology Laboratory,
Department of Environmental Science and En-
gineering, Indian Institute of Technology (ISM),
Dhanbad, Jharkhand, India
Archana S. Rao School of Basic and Applied Sci-
ences, Dayananda Sagar University, Bangalore,
India
xi
P.C. Rath ICAR-National Rice Research Insti-
tute, Cuttack, Odisha, India
Lopamudra Ray School of Biotechnology, Ka-
linga Institute of Industrial Technology (KIIT),
Bhubaneswar, Odisha, India; School of Law,
Kalinga Institute of Industrial Technology
(KIIT), Bhubaneswar, Odisha, India
Kiran K. Reddy ICAR-Directorate of Ground-
nut Research, Junagadh, Gujarat, India
Monire Mones Sardrodi Department of Plant
Breeding and Biotechnology, Faculty of Agri-
culture, University of Tabriz, Tabriz, Iran
Preeti Sengupta Amity Institute of Microbial
Technology, Amity University, Noida, Uttar
Pradesh, India
Efath Shahnaz Division of Plant Pathology,
Faculty of Horticulture, SKUAST-Kashmir, Sri-
nagar, J&K, India
Shilpi Sharma Department of Biochemical En-
gineering and Biotechnology, Indian Institute
of Technology Delhi, Hauz Khas, New Delhi,
India
Deepali Shukla Department of Biotechnology,
IPS Academy Indore, MP, India
Piyush Shukla Department of Rural Technolo-
gy, Guru Ghasidas Central University, Bilaspur,
CG, India
A.L. Singh ICAR-Directorate of Groundnut Re-
search, Junagadh, Gujarat, India
Poonam C. Singh Department of Plant microbe
interaction, CSIR-NBRI, Lucknow, UP, India
Prashant Kumar Singh Amity Institute of Mi-
crobial Technology, Amity University, Noida,
Uttar Pradesh, India
Bana Sravani Department of Plant Pathology
and Agricultural Microbiology, Post Graduate
Institute, Mahatma Phule Krishi Vidyapeet, Ra-
huri, Maharashtra, India
Ashmita Tandon Department of Plant Microbe
Interaction, CSIR-NBRI, Lucknow, UP, India
Stevenson Thabah Microbiology Laboratory,
Department of Biotechnology & Bioinformat-
ics, North-Eastern Hill University, Shillong,
Meghalaya, India
xii CONTRIBUTORS

Ajit Varma Amity Institute of Microbial
Technology, Amity University, Noida, Uttar
Pradesh, India
Ambarish S. Vidyarthi N.P.S Institute of Tech-
nology, GIC Campus, Pithoragarh, Uttara-
khand, India
M.K. Yadav Department of Plant Pathology,
Janta Collage, Bakewar, Etawah, UP, India
Manoj K. Yadav ICAR-National Rice Research
Institute, Cuttack, Odisha, India
 About the Editors
Professor Harikesh
Bahadur Singh is Distin-
guished ­Professor, Depart-
ment of Biotechnology,
GLA University, Mathura,
India. He has served as
Professor & Head, Depart-
ment of Mycology and
Plant Pathology at Institute of Agricultural
Sciences, Banaras Hindu University, India.
His major research focus is on bioinoculats,
biological control of plant pathogens and
nano-biotechnology. Prof. Singh has been
decorated with several prestigious National
awards and honors for his scientific contri-
butions in the field of organic farming and
translational agriculture, notable being CSIR
Technology Prize for Biological Sciences,
M.S. Swaminathan award by Society for
Plant Research, Vigyan Bharti Award, Prof.
V.P. Bhide Memorial Award, Society for Plant
Research, and many more. To his credit, he
has 20 U.S. patents which have been success-
fully transferred for commercial production
of biopesticides to several industrial houses
in India. He has been able to add more than
300 publications and 31 books Google Scholar
Citations- 10074; H-index- 52; i10- 182.
xiii
Dr. Anukool Vaishnav is
working as a Post-
Doctoral Scientist (Swiss
excellence fellow) at Uni-
versity of Zurich, Switzer-
land. He is also associated
with Department of Bio-
technology, GLA Univer-
sity, India as an Assistant
Professor. He has operated SERB-National
Postdoctoral Fellowship (NPDF) as Principal
Investigator at Department of Mycology and
Plant Pathology, Institute of Agricultural Sci-
ences, Banaras Hindu University, Varanasi,
India. He has >8 years of research experience
in plant-microbe interaction field. His research
is mainly focused on microbial mediated
plant protection against biotic and abiotic
stress, characterization of signaling molecules
and secondary metabolites (soluble and
VOCs) in plant holobionts. As an active
researcher, he has published more than
24 research and review articles along with
15 book chapters for reputed journals and
edited books. He has filed 9 Indian patents in
association with his research group. He is
editor of 2 Springer published books. He is an
editorial member of Current Genomics
Journal published by Bentham Science. He
has been awarded Swiss Excellence Post Doc-
toral Scholarship and several Young Scientist
Awards from different agencies.
 Preface
Zero hunger is a complicated task for scien-
tific community under this erratic climate
conditions, rapid population growth, and
with limited arable land. In the last two
years (2020 & 2021), the globe is encoun-
tered with the pandemic of Covid-19. This
outbreak causes declines and stagnation of
food trading and transportation between
nations that again affect global food supply.
To match with food demand, it is necessary
to apply such approaches that can increase
crop productivity and yield. The intensive
use of agrochemicals (fertilizers and pes-
ticides) has led to severe consequences,
including the deterioration of soil and the
environment. Increasing soil pollution and
adverse effects of agrochemicals on human
and animals built a pressure on agricul-
tural scientists to find alternate eco-friendly
approaches. In this context, application of
agriculturally important microorganisms
(AIMs) with the genera of Bacillus, Pseu-
domonas, Rhizobium, Burkholderia, Arhtobac-
ter, Trichoderma and Mycorrhiza spp. for the
purpose of biofertilizer and biopesticides is
a sustainable and eco-friendly alternative
of agrochemicals. The most consensual and
recent definition of these microbes states
“Plant Biostimulants”, which can be directly
applied to plants, seeds, and soil for sustain-
able agriculture production. These biostimu-
lants are part of a wide unknown microbial
diversity, constituting the rhizospheric, epi-
phytic, or endophytic microbiota, which are
xv
on the way to being cultured and preserved
at in-vitro conditions. This is an emerging
area in agrofarming, where a lot of evidences
demonstrate the potential of several AIMs in
promotion of plant growth and productivity
and also in disease management. However,
the mechanisms behind their efficacy and
interaction with soil biota and host plant
are still matter of discussion. The beneficial
microbes improve soil fertility and promote
plant growth through direct and indirect
processes. In direct process, these microbes
produce plant growth promoting hormones
and solubilize inorganic nutrients into the
soil that increase nutrient uptake efficiency
of plants leading to better growth and pro-
ductivity. Under indirect process, AIMs
work as biocontrol agent for different pests
and reduce pathogenicity in plants. There-
fore, these beneficial microbes can be used
as biofertilizers and biopesticides for cleaner
and healthier agricultural practices.
This book has reviewed the recent studies
on different agricultural important microbes
and emphasized on their future research
and application in agriculture. The chap-
ters contributed by prominent scientists will
be a ready source of information for young
researchers, industrialist and progressive
farmers.
Harikesh Bahadur Singh, Mathura, India
Anukool Vaishnav, Zürich, Switzerland
 C H A P T E R

1
Role of microorganism as new generation
plant bio-stimulants: An assessment
Deepali Shuklaa, Piyush Shuklab, Ashmita Tandonc, Poonam C.
Singhc, Jayandra Kumar Johric
aDepartment
of Biotechnology, IPS Academy Indore, MP, India, bDepartment of Rural
Technology, Guru Ghasidas Central University, Bilaspur, CG, India, cDepartment of Plant
Microbe Interaction, CSIR-NBRI, Lucknow, UP, India

1.1 Background

World’s largest population are dependent on plants or crop for their food requirements. For
the sustainable development of population, it is very essential that production of plants should
be raised more and more every time. In this context “Green revolution” played a vital role for
the production of rice, corn and wheat crop varieties. Moreover, different world governments
and organizations makes indispensable efforts to work on multiplication on the production
of crop to ensure the food provision (Abbott et al., 2018; Ortíz-Castro et al., 2012; Roberts and
Tasistro, 2012).
Studies suggest that for the increased productivity of crop, the role of nutrients like nitro-
gen, phosphorus, minerals and vitamins along with the plant health is very important. To
fulfilling demand of enhance crop productivity, in past decades chemical fertilizers market
were continuously growing globally (Singh et al., 2016). Intensive application of phosphorus
and nitrogen base chemical leads to a massive depletion of soil and water resources (Den
Herder et al., 2010), and it also cause global crisis of pollution. Enormous use of chemicals
is very harmful to human and environment too (Carvalho, 2017).
Other than use of chemical fertilizers for better crop yield climate change, emerging plant
pathogens, drought and stress is also a major challenge that affect agroecosystem (Rosenz-
weig and Tubiello, 2007). For surpass crop production, it is necessary to combat for all these
environmental hazards caused by the use of extensive chemical fertilizers and other climate,
plant disease and stress related problems for this some protocol and rules were established
for agropractices worldwide (Gomiero et al., 2011). Bio-stimulants gain popularity as addi-
tives to fertilizers which increase the efficacy of fertilizers by stimulating physiology of plant

New and Future Developments in Microbial Biotechnology and Bioengineering.

DOI: https://doi.org/10.1016/B978-0-323-85163-3.00016-8 1  Copyright © 2022 Elsevier Inc. All rights reserved.
2 1. Role of microorganism as new generation plant bio-stimulants: An assessment

and in current time microbial based bio-stimulant plays pivotal role in agribusiness (Backer
et al., 2018) and (Le Mire et al., 2016).

1.2 Introduction of plant bio-stimulants

Bio-stimulants can’t be precise in one function or one definition, it consists wide nature, they
act as additives to the fertilizers which stimulates/enhance the nutrient assimilation capacity,
growth promotion and increment of stress tolerating capacity of plants (Ricci et al., 2019).
Plant bio-stimulants are made up of any type of substance like enzymes, proteins, small
peptides, micronutrient, amino acids, plant and animal waste, plants extracts, microorganism
and microorganism derived products (Ugolini et al., 2015) and (Colla et al., 2017). Bio-
stimulants help in plant development and growth improvement by increment in seed ger-
mination in both above side or below side of plant, content of chlorophyll, size and number
of flower and fruits (Hermosa et al., 2012; Mendoza-Mendoza et al., 2018; Przybysz et al.,
2014; Ruzzi and Aroca, 2015) and (Drobek et al., 2019). Plant bio-stimulant can make the root
modification by which area of absorption are increased that lead to improvement in nutrient
uptake and translocation, it helps the efficient use of micronutrient attribute for enhancing
the plant biomass (Halpern et al., 2015; Samolski et al., 2012) and (Bargaz et al., 2018). Micro-
bial Plant Bio-stimulants are made up with varieties of microorganisms, they enhance the
growth, development adaptivity of different biotic and abiotic stress of plant and enhance
the crop productivity. Microbial Bio-stimulants are the new future in sustainable agriculture
practices (Alori et al., 2017) and (Rajput et al., 2019).

1.3 Basic mechanism of bio-stimulants

Main function of bio-stimulants is to improve productivity of plants by increasing the assimi-

lation of nutrient like C, N, S, P and other minerals (Du Jardin, 2015) and (Rouphael et al.,
2017), they also help to improve photosynthesis, stress toleration and mediate ion transport
also (Khan et al., 2009; Shukla et al., 2019) and (Zhu et al., 2019). Some reports also suggest
that bio-stimulants enhance production of various enzymes, proteins, amino acids, carbohy-
drates, pigments and phenolic compounds in plants (Parađiković et al., 2011) and (Bulgari
et al., 2017). Bio-stimulants also helps in reduction of reactive oxygen species which induce
stress and activates the antioxidant defense system of plant (Ertani et al., 2013) and (Yildiz-
tugay et al., 2019). Molecular studies suggest that some small molecular weight compounds
of bio-stimulants are participate in signaling events and influence the metabolic process of
plant (Saa et al., 2015) and (Trevisan et al., 2010).

1.4 Sources of plant bio-stimulants

Bio-stimulants may be made up of any chemicals, organic substance, plant extract and
microbes itself. Any substance to work as bio-stimulator that can help to improve the condi-
tion of crop, without creating any type of harm to the agroecosystem (Le Mire et al., 2016).

New and future developments in microbial biotechnology and bioengineering

 1.5 Microbes as plant bio-stimulant 3

In former agriculture practices, some organic compounds viz, humic acid and fulvic acid are
majorly used as chemical bio-stimulants (Canellas et al., 2015). Naturally occurring chemicals
like protein hydrolases, and phenols are served as natural bio-stimulants (Canellas et al.,
2015; Du Jardin, 2015; Pereira et al., 2019) and (Roomi et al., 2018). Vitamins, chitosan and
chitin are also in practice as plant bio-stimulants (Caradonia et al., 2019). After organic com-
pounds, plant and animal based organic substances were used as plant bio-stimulants, for
example naturally occurring row plant material, plant extracts, several type of fruits waste,
pulses, algal material (Battacharyya et al., 2015; Colla et al., 2015; Khan et al., 2009; Ugolini
et al., 2015). Organic, extract of different plant like rosemary oil extract increase the mass of
root by stimulating the nutrient uptake (Souri and Bakhtiarizade, 2019). Plant based other
bio-stimulant are mainly work as enzyme hydrolysis, these peptides substance or protein
hydrolysates stimulate growth of plant (Polo and Mata, 2018). Some plant bio-stimulants
preparation is made up with the mixture of plant and animal products that include small
peptides, hydrolase enzymes also some acids and alkaline products in animal waste, feather,
bone meals, collagens and casein (Colla et al., 2015) and (Ugolini et al., 2015). Several algae
or algal preparation are also work as bil-ostimulants and they show positive effect on many
types of crops (Godlewska et al., 2016) and (Amatussi et al., 2020). The preparation of organic
bio-stimulants is actually a fermentation process that can reduce the use of massive fertilizer
so it is eco-friendly option as well.
Microbial bio-stimulants are able to change the microflora of soil and plant by which
growth of plant/crop accelerate (Colla et al., 2015; Kannan, 2019). Microbial plant bio-
stimulant improves metabolic efficacy of plant so they brought many beneficial changes in
crop yield. Microbial plant bio-stimulants also improve the quality, shelf life, stress tolerating
capacity and disease resistance ability of plants (El-Mageed et al., 2017; Ma, 2019; Van Oosten
et al., 2017) and (Bulgari et al., 2017). In modern era of agroecosystem instead of conventional
strategies, microbial bio-stimulant get a popularity for their convenient preparation, small
inoculum size and eco-friendly nature (Arora et al., 2018) and (Verma et al., 2020).

1.5 Microbes as plant bio-stimulant

Agriculture faces several challenges, viz. massive use of chemicals, low crop yield and new
emerging virulent pathogens. For sustainable agriculture system it requires to minimizing
the use of chemical or toxic material, which are hazardous for agro environment and human
too (Keswani et al., 2014). Several factors of plant like root architect, p and n uptake, strengthen
natural immune system of plant (Péret et al., 2011), microbes have ability to change plant
root architect by changing the rhizospheric microflora which lead to more uptake of nutri-
ents, All these requirement suggesting foster by microbial based bio-stimulants (Abbott et al.,
2018; Berg, 2009; Calvo et al., 2014; Rouphael et al., 2017). Fig. 1.1, shows role of Microbes as
plant biostmulant.
In new era of modern agro practices, microorganism became vastly used plant bio-
stimulants also known as plant strengtheners and plant conditioner (La Torre et al., 2016).
Microorganism stimulate the growth of plant/crop in number of ways throughout the life
cycle from germination to maturity by enhancing the nutrient assimilation ability of plant.
Plant bio-stimulants can be applied on crop in many ways they directly apply on plant or

New and future developments in microbial biotechnology and bioengineering

 4

Agriculture Challenges Use of microbes as biostimulants Microbial Biostimulants

High crop yield

Low Crop Yield

Biotic Stress Fungus Induce SAR and SAR

Folliar application

Increase growth hormone production

Bacteria
Abiotic Stress

Enhance Stress toleration

Soil application
Microbial
Consortia
Poor Nutrition
Increase nutrient uptake ability

Root Application
Chemical Fertilizers
1. Role of microorganism as new generation plant bio-stimulants: An assessment

New and future developments in microbial biotechnology and bioengineering

Ecofriendly

FIG. 1.1 Effect of Microbes based bio-stimulants in plant/crop and its yield.
 1.5 Microbes as plant bio-stimulant 5

can be applied on seed. Microbial plant bio-stimulants are consider as safe for human, plants
and livestock. PGP microorganism also known as plant probiotics, and their bio-stimulants
activity plays a major role in world agro industries (Chen et al., 2003) and (Saa et al., 2015).
Microbial consortia as plant bio-stimulants are functions as agricultural of plant probiotics
(Kong et al., 2018; Wallenstein, 2017; Woo and Pepe, 2018) and (Dubey et al., 2019). Microbial
based bio-stimulants are being used in agriculture in early practice i.e. Rhizobium in legumes,
and currently it was used as commercial formulation for the improved or better nutrition for
the crop. There are several beneficial effects of microbes toward plants which are dependent
on chemical and nutritional signaling and soil and climate factors. As research reports that
sugars, acids and other compound secreted by plant root were affect the rhizospheric micro-
bial community leads to beneficial relation between plant and rhizospheric microbes
(Ortiz-Castro et al., 2017). Symbiotic relation developed between crop and soil microflora
includes PGPR which considers as natural bio-stimulants (Prasad et al., 2015). As compare
to conventional or organic farming microbial bio-stimulants are demanding in agriculture.
In continuation of this technology a new approach “Rhizosphereic engineering” is proposed
which is effective. Bio-stimulants are the natural preparation of substance or microbes that
support the plant growth/crop yield in eco-friendly aspect for the environment. Following
microbe are being used as microbes Phyto stimulant. Table 1.1 Shows some microbes and
their bio-stimulants activities.

1.5.1 Fungi as bio-stimulants

Plant growth promoting fungi are mostly use in agriculture from earlier time commonly
documented PGP fungi is mycorrhizal fungi which works as root biotroph (Giovannini et al.,
2020). Mycorrhiza mainly involve in gaseous exchange, and augment the absorption capacity
of roots, they also counteract towards negative effects of biotic and abiotic stress of plant.
Popularly used mycorrhizal fungi as bio-stimulants is Glomus intrarradicus, Funneliformis or
Rhizophagus, Gigaspora, and Laccaria (Colla et al., 2015).
Trichoderma are the most successful fungus used in multi benefits for plants worldwide as
plant bio-stimulants (Harman et al., 2004; López-Bucio et al., 2015; Woo et al., 2006) and (Vinci
et al., 2018). They are not only act as plant growth promoting microbes but also work against
phytopathogens. Several Trichoderma strains documented as effective biofertilizers, biopes-
ticides, biostimmulants and bioenhancer of crop. i.e. Trichoderma atroviridae Trichoderma reesei
and Heteroconium chaetospira (Colla et al., 2015; Du Jardin, 2015; Fiorentina et al., 2018; Gaiero
et al., 2013; Oancea et al., 2016; Woo et al., 2006) and (Fernando et al., 2018). Role of Tricho-
derma in plant growth promoting effect is to solubilisation of micronutrient and phosphate
(Vinci et al., 2018), It also mediate the release of secondary metabolite and Siderophores
(Lucini et al., 2019). Mechanism behind Trichoderma mediated Phyto stimulation involve
multistage communication with root and shoot system of plant. Trichoderma release auxin,
ethylene, small peptides, volatiles substance and other metabolites, which trigger the nutrient
uptake capacity and root branching so that plant growth and yield get boost (Contreras-
Cornejo et al., 2015) and (Poveda et al., 2019). Trichoderma sp. reported production of several
metabolic products including peptides secondary metabolites, cell wall degrading enzymes,
(Keswani et al., 2014) and (Ruocco et al., 2015) these are bio active compounds they affect
plant response to other microorganism by which plant defense mechanism is improved

New and future developments in microbial biotechnology and bioengineering

 6

TABLE 1.1 Bio-stimulants activity of different microorganism on various crop.

Microorganism Stimulation Activity Crop Reference
Trichoderma sp., Tricho- Bioenhancer of crop, resistance in biotic and abiotic Leafy Vegetables, Fruits, (Lorito and Woo, 2015), (López-Bucio
derma atroviride, Tricho- stress, Increase the synthesis of Hormones, Increase Cereals et al., 2015) and (Fiorentina et al.,
derma reesei. nutrient uptake capacity. 2018)
Heteroconium chaetospira Resistance Induction by production of, Jasmonic acid, Club root (Alfianny et al., 2017; Lahlali et al.,
Ethylene and Indole acetic acid. 2014)
Glomus intraradices Increase phosphate solubilisation, and biological Legumes (Rouphael et al., 2017b)
nitrogen fixation, and vitamins and mineral secretion.
Arthrobacter spp. Better Yield, Tolerance to biotic stress. Horticulture crop (Drobek et al., 2019)
Enterobacter spp. Better Yield, Tolerance to biotic stress, stimulation of Horticulture crop, beans (Galvão et al., 2019)
hormone synthesis.
Acinetobacter spp. Better Yield, Tolerance to biotic stress, stimulation of Vegetables and legumes (Calvo et al., 2014)
hormone synthesis.
Pseudomonas spp. Better Yield, Tolerance to biotic stress, stimulation of Vegetables and legumes (He et al., 2019)
hormone synthesis.
Bacillus spp. Better Yield, Tolerance to biotic stress, stimulation of Vegetables and Fruits (Kesaulya et al., 2018)
hormone synthesis.
Ochrobactrum spp. Better Yield, Tolerance to biotic stress, stimulation of Vegetables and legumes (Alfianny et al., 2017)
hormone synthesis.
Rhodococcus spp. Better Yield, Tolerance to biotic stress, stimulation of Vegetables and legumes (Cirou et al., 2012)
hormone synthesis.
Endophyte+ Fumic Acid Enhance Yield, Increase nutrient uptake. Sugarcane (Da Silva et al., 2017)
Trichoderma species +Ba- Increase the solubilisation of mineral, toleration if Grains, pulses (Oancea et al., 2016)
cillus sp. biotic and abiotic stress.
Trichoderma + Glomus Sp. have also been evaluated for their growth promoting Vegetables (Colla et al., 2015)
activity.
Trichoderma and Rhizos- Increase the N, uptake and translocation. Fruits and vegetables (Fiorentina et al., 2018)
pheric bacteria
1. Role of microorganism as new generation plant bio-stimulants: An assessment

Bacillus polymyxa and T. Reduce toxin Enhance Growth Grains (Lagogianni and Tsitsigiannis, 2019)

New and future developments in microbial biotechnology and bioengineering

harzianum
Rhizobium+Bacillus and Enhance mineral absorption, better Yield. Grains and vegetables (Rajput et al., 2019)
Trichoderma was ap-
plied to, resulting in
Pseudomonas sp. +Azos- Stimulation of defence and increase nutrients uptake. Wheat and other grains (Nguyen et al., 2014)
pirillum
 1.5 Microbes as plant bio-stimulant 7

(Vinale et al., 2008) and (Lombardi et al., 2018), It was reports that ACC deaminize and IAA
of Trichoderma regulate/stimulate the plant growth and defense (Hermosa et al., 2012).
Recent proteomic studies suggest that mitogen activated protein kinase is activated by Tricho-
derma which is transcription factor and DNA processing protein that represent formulation
of efficient product. Trichoderma also activate priming state (alert state) in plant by which
plant response will ready for pathogen attack this work as Systemic acquire resistance (SAR)
or Induce systemic resistance (ISR) (Hossain et al., 2017) and (Manganiello et al., 2018). When
live bio-stimulants are use either solid or liquid form they should have safe for human,
animals and plants also. Trichoderma formulation is the best example of suitable and safe
bio-stimulants in both liquid and solid phase.

1.5.2 Bacteria as bio-stimulants

Several bacteria are reviewed as PGPM for their beneficial role in plant microbe interaction
and growth promotion of plant i.e. Rhizobzcteria, non-pathogenic Pseudomonas sp., Bacillus
sp., Azosprulum sp., Azotobacter sp. and Serretia sp. Bio-stimulants PGP bacteria, formerly
known as PGPR but now many genera other than Rhizobia also investigated as good plant
growth promoting activity. PGP microorganism are very influential for plant life, they stimu-
late many factors of plant life, like growth and development, nutrition and morphogenesis.
These bacteria also develop the toleration of biotic and abiotic stress for plant and generate
the response to the other microorganism towards their ecosystem (Berendsen et al., 2012;
Berg et al., 2014; Bhattacharyya and Jha, 2012; Gaiero et al., 2013; Omomowo and Babalola,
2019; Philippot et al., 2013; Vacheron et al., 2013). PGPR are able to improve nutritional avail-
ability in soil for plants so help to assimilation and uptake in plant nutrition (Berg et al., 2014;
Raaijmakers et al., 2009). The main bacteria used as bio-stimulants are Arthrobacter sp., Acino-
bacter sp., Bacillus sp., Ochrobactrum sp., Pseudomonas sp. and Rhodococcus sp. (Calvo et al.,
2009) and (Gaiero et al., 2013).
Interaction of bacteria with plant is common in all possibilities, negative as well as positive
(Ahmad et al., 2008). Bacteria as bio-stimulants, plays successful role toward plant in many
ways, like extension of bacterial population in soil outside the plant root and inside the plant,
that is served as rhizosphere or rhizoplane bacteria sometimes the association may be per-
manent or transient. Some bacteria also make association with plant via seeds they stimulate
plant growth by supply of nutrients by participation in biogeochemical cycles. These bio
stimulants also enhance in nutrient use efficacy in plants, disease resistance induction, modu-
lation of plant morphogenesis by plant growth regulator stimulations and also increasement
in abiotic stress tolerant in plants. In agricultural use two type of bacterial biostiumulants
are being used, mutualistic endosymbiont bacteria and mutualistic rhizosphere PGPRs.
Rhizobia is most investigated genera in the field of agriculture as biofertilizers. Rhizobium
and related taxa are mainly involved in mutualistic endosymbiont type and are commercial-
ized in many biofertilizer combinations by facilitating nutrient requirements of plant (Kour
et al., 2019).
Azotobacter is free living N2 fixing bacteria that are nutrient influential. Azotobacter
secrets many substances like Phyto hormones, vitamins mineral and anther substance which
are plant growth promoter and regulator/ or stimulator (Hariprasad and Niranjana, 2009)
and (Wani and Arshid, 2013). Azatobacter desiccation resistance cyst formation capacity are

New and future developments in microbial biotechnology and bioengineering

8 1. Role of microorganism as new generation plant bio-stimulants: An assessment

use as microbial inoculant in durable and stress toleration ability in various environment
(Berg et al., 2014; Inamdar et al., 2000; Vacheron et al., 2013) and (Viscardi et al., 2016). Aza-
tobacter also tolerate biotic and abiotic stress with other microbes in agroecosystem that’s
why it is very useful in various commercial biofertilizer (Babalola, 2010; Berendsen et al.,
2012; Gaiero et al., 2013; Philippot et al., 2013; Viscardi et al., 2016) and (Van Oosten et al.,
2018). Variety of commercial biofertilizer have Azotobacter active ingredient or association
of Azotobacter with other microbes like bacteria and actinomycetes in the formulation
(Parađiković et al., 2019).
Azotobacter stimulate synthesis of superoxide dismutase (SOD), Catalase (CAT) and other
plant hormone which influence the plant health (Glick, 2014). Azotobacter as bio-stimulants
are reported in many crops such as tomato, maize, rice, wheat and sorghum (Di Stasio et al.,
2018; Viscardi et al., 2016) and (Van Oosten et al., 2018).

1.5.3 Microbial consortia as bio-stimulants

Generally several microorganism can do these functions alone but sometimes there is more
than one microbe, which shows good results in plant bio stimulation with synergistic behav-
ior. Some times in microbial source more than one microbe can be used called as consortium,
but sometimes it creates technical difficulties for results consistencies (Arora and Mishra,
2016). In present days group of bio-stimulants either microbial consortia or mixture of
microbes and chemical or organic substance also made a success story in agroeconomics. In
bio-stimulants research there are vast scope in mixture of bio-stimulants. Report says that
synthetic bacteria and fungi consortia have the ability to establish novel microbial commu-
nity in soil that also help in plant bio-stimulants (Berg et al., 2014; Du Jardin, 2015; Lugten-
berg, 2015) and (Woo and Pepe, 2018). This was also observed that if consortia work, single
microbe is not capable to show PGP effect (Wargo and Hogan, 2006).
New study suggest that Several consortia of microorganism integrated with botanical and
other inorganic compound, various seaweed and other animal product also can be used as
very good plant bio-stimulants and can became next generation plant bio-stimulants (Dubey
et al., 2020; Fiorentina et al., 2018) and (Chouyia et al., 2020) like Trichoderma sp. and Azoto-
bacter sp. Synergistic effect of Microorganisms are play very important role in biocontrol
activities are also served as a successful bio-stimulants. There is also future possibility in that
mixture of bioactive compound of Trichoderma and compound of other microorganism or
other organic and inorganic compound can develop a possible new generation of
bio-stimulants.

1.6 Role of microbes in nutrient uptake/stimulation

It is well known that microbes have versatile enzyme machinery which can transform the
nutrient in soil. Microbes enhance nutrient assimilation capacity of plant that results the
better yield of crop or better development of plant among all nutrient transformation nitro-
gen and phosphorus transformation are the major one (Richardson et al., 2009). Microbes
play following important role in plant bio-stimulants.

New and future developments in microbial biotechnology and bioengineering

 1.7 Conclusions 9

1.6.1 Nitrogen fixation

Nitrogen fixation is the process of fix the atmospheric nitrogen by microorganism, this activ-
ity found in certain group of microorganisms, the first biological nitrogen fixation was
documented in Clostridium sp. from which nitrogenase were isolated. Microorganism able to
fix nitrogen plays important role as bio-stimulants. Nitrogen is essential component of protein
and available very low in soil so microbes make availability of free N to plant by which N2
gives more healthy crop yield (De Luca et al., 2020). Bacillus sp. mainly convert complex
substance of phosphorus and nitrogen into simpler form so that p and n available for plant
nutrition (Richardson et al., 2009).

1.6.2 Phosphate solubilisation

Phosphorous in soil make salt, so it is unavailable for plants utility. In order to make the
availability of phosphorus, microorganism are best approach because they produce some
acid or enzyme that can solubilize phosphate from soil and intake of phosphorus increase
for plant which rein invent the plant growth (Sharma et al., 2013).
Bacillus megatarium, Bacillus circulans, Bacillus subtilis, Pseudomonas sp. and Trichoderma sp.
are the microbes which have ability to solubilise phosphate, these microbes create more
availability of phosphorus in soil (Mishra et al., 2014; Sharma et al., 2014). The prominent
phosphate solubilizing bacteria are Bacillus and Pseudomonas sp. These are commonly related
to rhizospheres zone also, when the preparation of these bacteria inoculated on plant because
of availability of phosphorus plant shows improved growth. Bacillus sp. mainly convert
complex substance of phosphorus and nitrogen into simpler form so that p and n available
for plant nutrition. In plants, phosphate also involve in nucleic acid, phospholipid production
and ATP metabolism so it stimulates many metabolic pathway and availability of phospho-
rus make crop healthy (Udvardi and Poole, 2013).

1.6.3 Hormones and other secondary metabolite

Hormones also play important role in plant metabolism and growth. Microbe base bio-
stimulants regulate the hormone activity like IAA, Jasmonic acid etc. production of plant by
which they easily adapt harsh environment and grow in better way (De Pascale et al., 2017).
Secondary metabolites are the products of various primary metabolic pathways, these are
mainly amino acids, phenolic compounds, alkaloids, sulphur containing compounds etc.
(Aharoni and Galili, 2011). Most of bio-stimulants have composition of plant hormones or
hormone like substances, proteins, small peptides various sugars, vitamins and other benefi-
cial substances. Microbial bio-stimulants produce stress metabolites and hormones that leads
beneficial effects on plant responses (Sharma et al., 2014).

1.7 Conclusions

Currently in agriculture field it is well known that microorganisms play important role in
plant. In positive approach they stimulate plant growth as well as boost the plant immunity.
From the use of single microbial inoculant to consortia microbial inoculant gain popularity

New and future developments in microbial biotechnology and bioengineering

10 1. Role of microorganism as new generation plant bio-stimulants: An assessment

in agribusiness as they are chemical free, eco-friendly and cheap availability and small
amount. Microbial bio-stimulants also known as plant probiotics, which help to enhance
plant growth promoting activities of fertilizers is a new approach to agribusiness worldwide,
these approaches also known as “rhizosphere engineering”. Treatment of microbial bio-
stimulants in soil activate phosphate solubilisation, biological nitrogen fixation, phytohor-
mone, siderophore and exopolysaccharide production that leads to enhance plant growth
and protect plant/crop from different biotic and abiotic stress (Haider et al., 2012; Van Oosten
et al., 2017; Viscardi et al., 2016) and (Woo and Pepe, 2018). A vast number of positive reviews
are being examined in favor of plant bio-stimulants but there are some challenges also there
in microbe base bio-stimulants due to lack of knowledge, most of farmers are not using these.
Another factor is that the market requires bio-stimulants which are broad spectrum not crop
specific. Other challenge of bio-stimulants industry is that the microbial bio-stimulants which
are ecologically safe has fewer common data so contemporary bio-stimulants are in more
practice. A very small number of microbes base bio-stimulants actually claim their actual
mode of action, stability or efficacy in field or broad spectrum crop and their response accord-
ing to the environment (Khan et al., 2009), so it is very necessary to regulate the selection of
suitable type of bio-stimulants, and there should enormous need to develop continuous
research in the field of bio-stimulants. In microbe based plant bio-stimulants research should
be focus on the inoculant survival and also study should be done interaction of native rhizo-
sphere microbe and microbial bio-stimulants (Conant et al., 2017; Rouphael and Colla, 2018)
and (Bonanomi et al., 2018).

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New and future developments in microbial biotechnology and bioengineering

Another random document with
no related content on Scribd:
"Just now we met Mr. Manners, little Miss Una's father, and he tells us that he
has news of David!"

"Of David?"

"Yes, he says that David is well and prosperous!"

"Thank God for that!" the old man exclaimed fervently.

"And that he is coming to see us!" Mrs. Maple continued. "He may be here
any moment!"

"Let me go to meet him!" Granfer cried excitedly.

"No, father, not yet! There is more to tell! David has been living near us some
time, but he never came to see us because he was afraid you were angry and
bitter against him still; and father, he has a child of his own whom you already
dearly love! Oh, cannot you guess who our David is?"

The old man shook his head, and looked around him in painful bewilderment.
Suddenly Nellie gave a little cry of glad surprise.

"Oh, I know, I know!" she cried excitedly. "Uncle David is Mr. Manners, and
Una is our cousin!"

"Yes," Mrs. Maple replied, "you have guessed rightly, Nellie." And turning
again to Mr. Norris she said: "Una is your own grandchild, father; do you
understand now?"

Granfer made no response in words, but his full heart arose in a prayer of
thanksgiving to God. There was a brief silence, then the farmer beckoned to
the children to follow him, and together the three left the kitchen, while Mrs.
Maple hurried to the door and spoke to some one who was waiting without.

"I have told him, David," she said softly. "You can come in and see him now!"

Granfer turned his head quickly, and peered at the tall form that came to his
side with outstretched hands that sought his own.

"Father, forgive me!"

It was his son David's voice, and the old man trembled exceedingly, whilst his
quivering lips murmured the two words: "My son!"
Mrs. Maple stole gently away to join her husband and children, and to have a
good cry, because, as she said, she was so very, very glad.

Then, after an hour had passed, they all returned to the kitchen to find father
and son seated side by side talking quietly and happily.

"This is the one like you, David," Granfer said, calling Bessie to him. "I wonder
you never noticed the likeness yourself! Your little Una does not favour you in
the least!"

"No, she is like her mother, and I am glad she is!"

"Her mother must have been a very sweet woman, I am sure," Mrs. Maple
said.

"She was indeed. Her death was a great trouble to me; we had only been
married eighteen months when she died. My little daughter is very fond of you,
father."

"Yes, she is," the old man admitted, smiling with pleasure. "The first time we
met she asked if she might call me Granfer."

"Did she really? How strange!"

"And it was she who led me to pray for you, David: I never did till your child
suggested it! God bless her!"

"She is a dear little soul—my Una! Ah, she has looked forward joyfully to the
return of your son, never dreaming him to be her own father!"

"Have you not told her yet?" Mrs. Maple asked.

"No, but I shall do so to-morrow."

"Cannot you manage to bring her here to tea? It is father's eightieth birthday,
you know, and he will want you and Una to taste his birthday cake. Oh, you
must come, David!" Mrs. Maple said appealingly.

"I will certainly see if it can be managed in some way," he responded smiling.

"We will send the gig up to Coombe Villa to fetch you," the farmer offered
hospitably.

"Thank you; if you do, we will certainly come. Una will be delighted, I know."
"Shall we call you Uncle David now?" Bessie asked, looking up into her new-
found uncle's face, with shy, dark eyes.

"Yes, indeed you must, and Una is your cousin, remember. But my name is
really Manners," he added, turning to Granfer. "It was my wife's maiden name,
and on our marriage her father stipulated I should take his name on account
of some property which had to come to his daughter at his death. He died
seven years ago."

Mr. Manners remained some time longer, but at last he rose to leave, saying
that Una would wonder what had become of him; and after he had gone a
silence fell upon the little party in the farm-kitchen, which was broken only by
Mrs. Maple remarking:

"I feel as though I must be dreaming! I cannot realise that he is really David—
can you, father?"

"Yes, I recognised his voice in a moment. I had never seen him since he came
to live at Coombe Villa except in church, but if I had met him face to face, I
should have known him immediately," the old man declared, with conviction in
his tones. "God has been very merciful to me, and heard my prayers, and I am
happier to-night than I have been for years—indeed ever since David went
away."

"Will Uncle David show you the beautiful picture he is painting, Granfer?"
Bessie asked. "Oh, you will want to see it, won't you?"

"Yes, I shall," he acknowledged. "Do you remember, Bessie, when you said if
painting was his talent, it would have been wrong for him not to use it?"

"Of course I do, Granfer!"

"You were quite right, my dear, quite right!"

"Has God given you your heart's desire, Granfer?" Nellie questioned softly.

"Ay, that He has!"

"And now you will be so happy, won't you?" the little girl continued: "and you
shall have such a beautiful birthday, and Uncle David and Una will come to
tea, and we shall all have such a lovely time together! Oh, I wonder what Una
will say when she knows that you are really and truly her grandfather?"
"She will be very pleased, I feel sure," Mrs. Maple said, her face beaming with
happiness. "You children shall have a holiday from school to-morrow, and then
you will be able to help me get everything straight by the time our visitors
arrive!"

CHAPTER VIII
GRANFER'S EIGHTIETH BIRTHDAY

FAIR dawned the morning of Granfer's eightieth birthday. The sun rose behind
a gray mist which it quickly dispelled, and shone on a world decked with fresh
green fields, tender budding leaves, and myriads of flowers. Never during all
the eighty years of his long life had Granfer seen a more beautiful May
morning; never had his heart beat happier, or his soul been filled with a
greater joy, than to-day as he came downstairs to be greeted with good
wishes, loving kisses, and kindly looks from each member of the family in turn.

At Coombe Villa Mr. Manners was awake and up early. He went into the
garden and gathered a bunch of flowers for his little girl; then returned to the
house to wait till she should come downstairs. At last she entered the room,
looking a trifle pale still as a result of the shock of her fall, and limping in her
walk, but smiling and bright as ever.

"Oh, you dear old father!" she cried when she caught sight of the flowers. "I
know those are for me!"

She went up to him and put her arms around his neck, giving him a tender,
loving kiss.

"Has my darling slept well?" he enquired.

"I fell asleep the minute I was in bed, and never woke up till Nanny called me
just now," she answered.

"Is it not a lovely morning?"

"Beautiful. There has been a heavy dew during the night, and everything in
the garden is the fresher for it."

"It is Granfer's birthday," she reminded him. "I wonder if his son has come
home?"

"Yes, Una; he returned last night!"

"Oh!"

For a moment she said no more; her lips trembled with emotion, and her eyes
shone through a mist of tears. Presently she said simply:

"God has answered Granfer's prayers at last."

Her father kissed her again and again.

"Was Granfer very delighted to see him?" she asked. "How did you know,
father?"

Then he told her the whole story, how he himself was Granfer's son, and how
he had gone to Lowercoombe Farm the night before and become reconciled
to his father. She listened in silence, too amazed to utter a word, but there
was a glad light upon her face and joy in her tremulous smile.

"So you see, Una darling, Granfer is your grandfather as well as Nellie's and
Bessie's; and Mrs. Maple is my sister Mary, and your aunt," Mr. Manners said
in conclusion. "They are so pleased to think that you are related to them, and I
have promised that we will have tea at the farm this afternoon because it is
Granfer's birthday, and he wishes it."

"Oh!" cried Una again, with a little gasp of astonishment. "Oh, how wonderful!
Father, you only told me half the secret, after all! You never said you were
Granter's son! I am so glad you are! Oh, dear Granfer, how pleased and
happy he must be!"

"And I am happy too, Una, happier than I have been for many a long year. It
was you who told me my father had forgiven me and wished to see me again."

"Granfer loves you so much, father darling, and I am sure you love him, don't
you? Fancy Nellie and Bessie being my cousins! But I don't think I can care
for them any more than I do now, because I am really very fond of both of
them, and I'm sure I don't know which I like best!"
Una was full of excitement. After breakfast there was Nanny to be told the
wonderful news, and to the little girl's great astonishment she discovered that
her nurse was not so surprised as she had anticipated she would be.

"I have guessed your father was old Mr. Norris's son for some while now,"
Nanny confessed, "on account of different things I have heard Mrs. Maple say
about her brother, and by putting two and two together. I am glad it has come
out at last. Ah, Miss Una, this life is too short for folks who love each other to
be angry long; we ought to learn to forgive and forget!"

"Granfer is not angry now!" Una said quickly, fearful lest Nanny should not
have grasped that fact.

"I should think not! There, dear, I won't say a word against your grandfather,
for I believe the good Lord has really softened his heart; even to my eyes, he
doesn't look quite so grim as he used," and Nanny gave Una a kiss, adding
gently, "God does everything for the best, my dear, and He makes all right in
the end!"

At four o'clock the gig arrived from Lowercoombe Farm, driven by the farmer
himself, and with the faithful Rags in attendance.

"I hope you are going to take kindly to your new uncle?" Mr. Maple said with a
merry twinkle in his eye as he lifted Una in his arms to put her into the
conveyance.

For answer the little girl clasped him tightly round the neck, and, after pressing
a kiss on his bronzed cheek, answered promptly:

"Indeed, I love you very dearly already! You are so very kind!"

They drove off, Una seated between her father and the farmer, the latter
amusing her with an account of how the tame lamb was daily growing bolder,
so that they had great difficulty in keeping it out of the house.

"It's so tame that it follows my wife everywhere," he declared, "and she has to
shut it up on Sundays when we go to church, or we should never keep it at
home!"

In ten minutes they arrived at Lowercoombe Farm, where Una was carried
into the kitchen, and surrounded by her aunt, and grandfather, and the
children. They had so many questions to ask and to be answered, and made
so much of her, that she felt quite bewildered at first; but by-and-by she
noticed that Granfer was wearing the suit of clothes he usually wore on
Sundays, and that Mrs. Maple and Nellie and Bessie were attired in their best
gowns in honour of the day.

Presently, they had tea in the best parlour, which room was only used on great
occasions, or when there were visitors at the farm. Una sat between her father
and grandfather in a sort of dream-like happiness. In the centre of the table
was Granfer's birthday cake, which he cut himself, and every one declared to
be most delicious.

The old man and each member of the little party seemed merry and pleased.
Now and again Una met her father's eyes and smiled in answer to his
affectionate glance that mutely asked if she was happy and content. She was
both, though it seemed very strange to find herself and her father so much at
home at Lowercoombe Farm.

During the evening the little girl had a few words alone with her grandfather,
and took the opportunity to tell him how glad she was to know of the
relationship between them.

"God has been very good to me, my dear," he said gratefully. "He has given
me back my son."

"Yes," she answered, smiling brightly, "God is good." She took his hand in her
little, soft fingers, and looked tenderly into his aged face, as she added
lovingly:

"He has given me my Granfer too!"

 ONE CHRISTMAS TIME

CHAPTER I

CONCERNING A DOLL

IN AN AMBER-COLOURED GOWN

IT was a wretched evening, only a few days before the joyful Christmas
season. The weather was damp and chill, and the London streets were
slippery and comfortless. Pale, shivering forms sheltered themselves in every
conceivable nook which was safe, for a time at any rate, from the keen
scrutiny of the police; business men and women were wending their different
ways homewards from the City, and the theatres and other places of
amusement were not yet open. The shops were with enticing articles
displayed to the best advantage, and many a poor child stood wistfully gazing
at the fruit in the grocers' windows, so temptingly set out, as though to
purposely tantalize hungry eyes.

And then the toys! Wonderful inventions made for the children of the wealthy!
Engines worked by machinery! Dolls that opened and shut their eyes, and
even walked and talked! Noah's arks of marvellous workmanship, containing
every known animal on the face of the globe!

A young man, hurrying along, turned his head and glanced smilingly at a shop
window full of dolls of all sizes and conditions and prices, from the gorgeously
apparelled waxen bride-dollie in satin and orange blossom to her penny Dutch
sister with flat figure and nondescript features.

"That would be the place to buy a doll for Nellie!" exclaimed the young man,
as he came to a full stop and stood with his hands in his pockets, gazing at
the motley faces that seemed to stare at him unblinkingly with their glassy
eyes. "I suppose she would rather have a doll than anything else, although
she has so many already!"
He was a good-looking lad, a medical student, Jim Blewett by name, and
Nellie was the only child of his brother in Cornwall, and a great favourite with
her uncle.

"I think I could afford half-a-sovereign," he ruminated; "for that price it would
appear one can get a most desirable dollie!"

He was turning into the doorway of the shop when he espied a child at his
side, watching him with great interest, and he paused. She was a little girl of
about seven years old, with a pale, thin face, and large dark eyes. She drew
back when she saw he had observed her, and coloured. His shrewd glance
noted she was poorly though neatly clad, and that her toes had worn through
her boots, whilst her head was covered by an old sailor hat much too large for
her, and with a dilapidated brim.

"I suppose I must have been talking aloud," Jim Blewett thought; then nodded
encouragingly at the child, who responded with a smile.

The young man was not a Londoner; he was only studying at one of the
London hospitals, and looking forward to the day when, fully qualified, he
would be at liberty to practise his profession in the country. Brought up in a
small provincial town, where he had known all the inhabitants, at any rate by
sight, he could never understand the unconcern with which Londoners regard
those who cross their path. He was always picking up acquaintances in an
eccentric manner, as his fellow students declared, or mixing himself up in
other people's business.

"It would be a much more unhappy world than it is, if no one interfered with
what did not immediately affect himself!" Jim would retort good-naturedly. He
was certainly in disposition very unlike the priest and the Levite in the parable,
for he was always ready to go out of his way to assist any one; his desire was
to be neighbourly to all the world. The young man was a general favourite,
and though many of his acquaintances laughed at him, they could not help
admiring him for his open, manly Christianity.

"Well, little one," he said cheerily, "are you having a peep at the dolls?"

"Yes, sir," the little girl responded, in a slightly abashed tone.

"I suppose you have a doll of your own at home?" he proceeded to enquire.

The child shook her head, whilst a smile crossed her face, as though she was
amused at the thought. Then she turned to the window again and sighed.
After watching her a few moments in silence, Jim drew nearer and asked:

"If you had the money, which would you buy?" She glanced at him doubtfully,
being mistrustful of a stranger, but, reassured by his kind face, pointed to a
large rosy-cheeked doll, in a gaudy amber-coloured frock. Jim saw it was
ticketed half-a-crown.

"If I gave you that doll, would you be pleased?"

She looked at him hesitatingly, then drew back, the tears springing to her
eyes, her cheeks crimsoning.

"You're making game of me!" she cried.

"On my honour I am not! See here!" Jim seized the child's chill hand in his
warm clasp, and drew her into the brilliantly-lighted shop. "Will you please let
us see one of the dolls in the window?" he asked of the young woman who
came forward to serve him. "It is the one in the yellow dress we want. The one
marked half-a-crown."

In a minute the much-coveted doll was laid on the counter, and Jim turned to
his companion.

"Is that the one you would like?"

The little girl lifted her eyes to his smiling countenance, her face alternately
paling and flushing with excitement.

"Oh, sir!" she gasped. "Oh, sir! Do you really mean it?"

"Mean it? Of course I do! I'm going to give you a Christmas present because I
have a little niece about your age, and you remind me of her, and I know if she
was here she would want you to have this doll!"

"Oh!"

"I will put the doll in paper," said the young woman behind the counter.

"Perhaps you would rather take her as she is?" asked Jim. "Or shall the lady
wrap her up for you?"

"She might feel the cold!" the child answered, looking at the doll with longing
eyes.
"She might," he agreed laughingly. "We will have her put in paper, please."

The assistant turned aside, and in a minute brought forward a cardboard box,
into which she carefully laid the doll, then, after wrapping the box in paper and
securely fastening the parcel with a string, she handed it across the counter.

"There, my dear," she said, as the little girl took possession of her present,
"your doll will be perfectly safe now."

"Oh, thank you, ma'am!"

"Thank you," Jim said, as he paid his half-a-crown. "You are very good to take
so much trouble!"

"Not at all, sir! I am only too pleased!"

The young woman, who was weary with standing all day, and had been
feeling decidedly cross and disheartened, seemed considerably cheered by
the sale of the doll. She watched Jim and his companion leave the shop with
interested eyes.

"What an odd couple!" she thought. "Fancy him spending his money on that
street child! Well, he must have a kind heart!"

Meanwhile Jim Blewett was saying good-bye, and refusing to listen to the
thanks which the delighted little girl was trying to put into words.

"Run away home," he said, "and take care of your dollie. I hope she'll be a
good child, and give you no trouble!"

"Oh, sir! I can never, never thank you!"

"Never mind. I don't want thanks. Good-bye."

"Good-bye, sir!"

The child gave him a long look full of gratitude; then, clasping her treasure
closely in her arms, she darted down the street, and was soon lost in the
hurrying crowd of pedestrians.

"Poor little soul!" Jim thought. "I'm glad I was able to gratify her desire. Well,
Miss Nellie will not have such an expensive present as I intended, but she
won't mind, and I think I'll write and tell her of this little adventure; she will be
interested."
The young man hastened home, swinging along with easy strides, his
thoughts busy with his little niece in Cornwall and the child whom he had
rendered happy in his impulsive way. Arrived at his lodgings, he found his tea
awaiting him, and his landlady forgot her household cares as she answered
his cheery greeting.

"A dull evening, Mrs. Metherell," he remarked, as she brought in the tea-pot,
and he sat down to his frugal meal; "but I never mind the weather! We shall
soon have Christmas now, and I begin to feel quite Christmassy already!"

CHAPTER II
HOW THE DOLL WAS RECEIVED

IN THE BLUNDELL FAMILY

"OH, how my eyes ache!"

The speaker, a weary-looking woman, was seated stitching away by the light
of a single candle. She was a button-hole maker, so what wonder if her poor
eyes did ache! To make button-holes from early morning till late at night is no
easy task; but Mrs. Blundell was not usually a grumbler, and she rarely
complained. To-night she was very tired, and a fear that had haunted her for
months took strong hold upon her, and filled her soul with dismay. Supposing
there should be something really amiss with her eyes—more than weariness?
Supposing her precious sight should be really leaving her? She shuddered at
the thought, for she had two children to support, and, as things were,
existence was hard enough.

But Mrs. Blundell was one who always put a stout heart to a stiff hill. She had
been country bred, and had come to London as a wife ten years ago. Her
husband, a house painter by trade, had been led astray by evil companions,
and had taken to drink and gambling. The downward path is always a swift
one, and so it had been in John Blundell's case. When he had died, nearly two
years since, he had left his widow and two little girls totally unprovided for; and
Mrs. Blundell continued to work as a button-hole maker, as she had done
during her husband's lifetime, in order to supply those necessaries which were
so hard to provide.

"'As thy days, so shall thy strength be,' the poor woman had murmured to
herself over and over again when the weight of care thrown upon her would
have seemed unbearable except for that great promise. She had learnt to turn
to her Heavenly Father for assistance in time of trouble, and trusted in Him
with all her heart. But to-night she was wearied out, mentally and bodily; and
as she glanced round the garret that was home to her and her children she
shuddered at the thought that even this humble abode might not be theirs
much longer.

"Mother!"

The voice, weak and plaintive in tone, proceeded from a bed in a corner of the
room, where a little girl of about eight years of age was lying.

"Yes, my darling!"

The mother spoke in tender, caressing accents, which she strove to make
cheerful for her sick child's sake. Little Annie was always ill. She suffered from
a spinal complaint, and only Mrs. Blundell knew that it was the result of a fall
she had had from her father's arms when an infant. John Blundell had been
intoxicated when the accident had happened, and, though it had been a shock
to him at the time, he had soon recovered from his fright, although Annie had
never had a day's health since.

"Oh, mother, do put down your work, and rest your poor eyes!"

"Presently, my dear; I am not going to do much more to-night. Have you been
asleep, Annie?"

"Yes, mother. Where is Maggie?"

"I sent her out to get some thread an hour ago. She ought to be back by this
time."

"I expect she is looking at the shops. She was telling me this morning how
they were. Oh, how I wish I could see them!"

"I wish so too, Annie."

"It does seem hard not to be able to get about like Maggie. Oh, mother, why
are we so poor? Has God forgotten us, do you think?"

"Oh, hush, my dear! No! God has not forgotten us; that is impossible! All the
world may forget us, but not God!"

"But, mother, it's so hard to be poor at Christmas time!"

"Oh, Annie, don't say that! The joy of the Christmas season has nothing to do
with riches, although it must be pleasant to be able to give happiness to
others for Christ's sake. If we have no money to buy presents for those we
love, the love is in our hearts the same; and the angels' message was to the
whole world, rich and poor alike. Never mind our poverty, Annie, so long as
Jesus is with us. Have you forgotten how He was born in a stable, and cradled
in a manger, because His mother was of so little account that they could not
make room for her in the inn?"

"Oh, mother, I do remember, but—"

"He was poor all His life," Mrs. Blundell continued softly, "and His friends were
the working people. That thought has helped me to bear a great deal, for He
understands all our trials and sorrows."

"Still, I should like to have some money to buy presents for you and Maggie,
mother?"

"What would you give me, my dear?"

"A new gown, mother; it should be so warm and soft! I am not sure what I
would give Maggie! A pretty new hat, I think, for that old sailor hat of hers is
dreadfully shabby."

There was silence for a few minutes, then the sick child spoke again:

"Mother, I can't see the stars to-night; I expect it is raining."

Mrs. Blundell put down her work, and rising, went to the tiny window, and
looked out.

"It is very misty," she said. "I do wish Maggie would come! Where can she
be?"

"She is coming, mother!"

Annie's quick ears had told her truly, for in another minute the rickety door was
flung open, and a breathless little figure ran into the room, and stood panting
before her astonished mother and sister.

"Maggie!" exclaimed Mrs. Blundell, with mingled anxiety and reproof in her
voice. "Where have you been?"

For answer the child laughed—a clear, ringing laugh, full of pure enjoyment,
that echoed strangely through the miserable garret. Annie raised herself on
her elbow, her eyes open wide with amazement, whilst Mrs. Blundell pointed
to the parcel in Maggie's arms for an explanation.

"Oh!" the excited little girl cried at length. "You'll never guess what has
happened."

She laid the parcel on the bed, and bade Annie open it, then stood by,
somewhat impatiently watching the weak, tremulous fingers as they fumbled
with the string. The cover was removed from the box at last, and the doll in the
bright amber gown lay revealed.

For a moment there was an awed silence; then:

"Where did she come from, Maggie? Who is she for?"

"She is for you, Annie," Maggie answered brightly, "for your very own! A
Christmas present! A gentleman gave her to me, and I ran as fast as ever I
could to bring her home to you! You remember my telling you yesterday about
that shop where there was a big window full of dolls? Well, I was looking in,
and the gentleman asked me which doll I would like if I had the money to buy
her; and then, when I told him, he took me into the shop and gave her to me!"

"Oh, Maggie!"

"And the lady in the shop put her in this box because she should not get
damp," the excited child continued, "and then I ran home as fast as I could!"

"What a kind gentleman he must be!" cried Annie. "I wonder what made him
do it! I think God must have told him!"

"I shouldn't wonder," Maggie agreed. "How do you like her, Annie? You
haven't touched her yet!"

"She is so pretty, and her dress is so grand," the sick child answered in awe-
struck tones. "I never saw such a lovely doll before."
"She is your very own, Annie."

"Oh, I don't like to take her from you, Maggie; the gentleman meant you to
have her!"

"I would rather give her to you!"

Taking the doll carefully from the box, Annie placed her in her sister's arms,
whilst Mrs. Blundell stood by, watching the children with tears in her eyes. She
was pleased to see Maggie acting so unselfishly, for she well knew that in
giving up her doll the child was making no slight sacrifice.

"See what lovely pink cheeks she has!" cried Annie. "And, oh, how blue her
eyes are! Oh, you beautiful creature!"

"I knew you would like her," Maggie remarked complacently. "I was longing to
be to able buy her for you when the gentleman spoke to me."

"Was he an old gentleman, Maggie?"

"Oh, no—quite young."

"He must have a kind heart," Mrs. Blundell said gratefully. "The doll will be
quite a companion for Annie when you are at school, Maggie, and I am busy."

It was a strange scene in that humble garret home—a scene full of pathos and
tender human nature. The sick child with the gaudily dressed doll clasped in
her frail arms; her sister, her face radiant with happiness; and the careworn
mother looking on with eyes that smiled through a mist of tears.

It was Mrs. Blundell who broke the silence in words that came straight from a
heart full of thankfulness and gratitude:

"There, children! You have had a beautiful Christmas present! You have no
idea who the kind gentleman was, my dear? No. Well, God bless him,
whoever he may be!"
 CHAPTER III

CONCERNING JIM BLEWETT AND HIS LANDLADY

THE following morning the children awoke early, and the chill winter's dawn
found them busily discussing the marvellous attractions of the wonderful doll.
Annie was so excited that she could scarcely eat a mouthful of breakfast, and
Maggie was nearly as bad. It was certainly not an inviting meal, being
composed of a little weak tea and slices of bread and dripping; but the
children ate so sparingly. Annie, posted up in bed, looked better than usual.
She was a pretty child really, but sickness had made her wan, and had
sharpened her features till she seemed all nose and eyes.

"What shall we call her?" she asked, pointing to the doll lying on the
counterpane by her side. "She must have a name."

"Call her 'Rose,'" suggested Mrs. Blundell; "or do you want something that
sounds grander?"

"I don't know," doubtfully. "What do you think, Maggie?"

"I think 'Rose' would do splendidly. She has such rosy cheeks, hasn't she?"

"Yes. And such lovely hair and eyes! She is beautiful! We will call her 'Rose,'"
and the little invalid looked at the doll with admiring eyes, and gently
smoothed the amber gown.

When breakfast was over Maggie started for school, and Mrs. Blundell was
obliged to go out to take her work to the business-house that employed her;
and for the first time in her life the time Annie spent alone did not seem very
long. She lay back in bed, feeling perfectly happy and contented, talking to her
doll, which she held in her weak arms, and every now and again pressing
tender kisses on the blooming cheeks.

"Let us 'make believe,'" she whispered. "We are in a palace, a beautiful palace
made of white marble, and the walls are shining with diamonds, and there is a
grand feast for every one, and there are flowers everywhere! The King is
having a party, and nobody is cold or hungry, because the King is so good and
wise, he won't allow people to be unhappy or want for anything. It is warm,
and oh, so comfortable! The King has asked us to sit by the fire with a lot of
other little children, and we can feel the heat!"

The child paused, and shivered involuntarily, awakening suddenly to the

reality; but in a minute she smiled, and continued to "make believe."

Meanwhile, the wintry sunshine was peeping through the tiny window; the mist
was clearing, and in the streets people were remarking that there was a
promise of a real old-fashioned Christmas.

Jim Blewett as he sat at his breakfast table looked at the sunshine, and
smiled.

"The weather is going to change," he remarked to his landlady as she placed

his fried bacon in front of him; indeed, "it has changed already. There's a more
cheerful outlook this morning."

"Yes," she assented, "I expect we shall have a spell of real cold now. God help
the poor folk if we do!"

"It will be healthier than all the damp we've been having, Mrs. Metherell."

"Maybe, but it'll be a deal more trying for the poor. You don't know London like
I do, sir, or you'd know that!"

"I was bred in the country, thank God; and in the small town where I was
brought up the poorest never lacked for fuel, I am sure. My father was the
Vicar—he died several years ago—but in his young days, he had had some
experience of London life, as he had held a curacy in the East End. He
accepted a living in Cornwall when I was a baby, so my knowledge of London
is built on what I heard from him, and my own two years' sojourn here."

"I was born and bred in London," Mrs. Metherell declared, "and I've an
affection for the place, though they do call it modern Babylon. I don't suppose
people are worse here than in the country. There's a deal of wickedness done
in London, I must confess, but there's a deal of goodness too! For my part, I
love the bustle, the continual movement, the life! It seems to me country folk
are never properly alive!"

"I suppose all Londoners think that!" Jim replied laughing, as he looked at his
landlady's good-humoured face. "I must acknowledge that you always strike
me as being very much alive, and you say you're a real Londoner. You do not
let the grass grow under your feet, Mrs. Metherell."
Jim knew no woman could possibly work harder than his landlady. She was at
it early in the morning, and late at night; yet she was always bright and
cheerful.

Mrs. Metherell was a little woman with a tip-tilted nose, a pair of honest gray
eyes, and a wide mouth which was redeemed from ugliness by a beautiful
smile. Her figure was spare, and she stooped slightly, as though she had been
accustomed to carrying heavy weights, but she was quick in her movements,
and her tongue was quite as nimble. Left a widow at thirty, she had, by means
of this lodging-house in a quiet side street, contrived to bring up three
children, and put them out in the world. They all had homes of their own now;
but their mother kept on the lodging-house, and her cheerful countenance
grew brighter still as the years passed on, and she found herself in easier
circumstances.

Jim Blewett had lodged with Mrs. Metherell for the last two years. They had
taken to each other at the beginning of their acquaintanceship, for, strange
though it may appear, they were congenial spirits. The toil-worn Londoner and
the country lad had much in common; they met on the ground of their wide-
hearted Christianity. Mrs. Metherell often lingered thus for a few minutes'
conversation, and Jim, being of a decidedly sociable disposition, always
encouraged her to talk.

"You are not thinking of going home for Christmas, then, sir?" she asked.

Jim shook his head, whilst a shadow crossed his face.

"No," he said, "although my brother's wife has written and asked me to come,
and my little niece Nellie sent a message to say it wouldn't be like Christmas if
I was not there! But I have made up my mind not to go down till Easter. I want
to work, and a break now would unsettle me, I know. If my mother and father
were alive it would be different!"

"Ah! This season brings its sad memories to many a heart," Mrs. Metherell
remarked, "but they cannot take away from the joy. The house will be well-
nigh empty this week, as most of my lodgers will be away. I am going to give a
party on my own account, sir!"

There was a twinkle in Mrs. Metherell's eyes as she spoke, which Jim was
quick to notice.

"I hope you are going to invite me," he said, smiling; then, seeing Mrs.
Metherell looked a little doubtful, "You surely won't leave me out in the cold!"
"It's a children's party, sir."

"So much the better! They are ever so much jollier than grown-up people's
parties! I will help you amuse the children!"

"They are a few I know who will not be very likely to have anything done for
their pleasure at home. I am going to ask them to come on Christmas Eve
from four to eight. I shall give them a good tea—poor little souls! And I mean
to dress up a Christmas tree for the occasion!"

"I shall insist on being present, Mrs. Metherell; and I'll help you dress the tree!"

A smile of gratification spread over the landlady's face as she answered:

"I am sure I shall be very glad of your assistance, sir. It's not much I'm able to
do for my fellow creatures; but now, at Christmas, I think one ought to make a
little extra effort to try to make others happy. It always seems to me Christmas
is the children's festival especially, and I should like to think I was able to
make some of His little ones glad, for His sake."

"Yes," Jim agreed. "I remember—oh! As long ago as I can remember anything
—the excitement there used to be at home when this season drew near, and
how my father used to remind us children that we must never forget in the
midst of all the festivity the cause of our rejoicing."

"He was quite right, sir."

"Indeed, he was. Well, I hope you and I shall have a happy Christmas."

His thoughts flew to the child to whom he had given the doll the night before,
but he made no mention of the matter to his landlady. And after a few more
words she retired downstairs, and Jim turned to his breakfast.

CHAPTER IV
MAGGIE IS INVITED TO A PARTY