Nanobiotechnology Applications in Plant Protection: Volume 2 [1st ed. 2019] 978-3-030-13295-8, 978-3-030-13296-5

Table of contents :
Front Matter ....Pages i-xiv
Intellectual Property Rights in Nano-biopesticides (Prabuddha Ganguli)....Pages 1-17
Application of Nanomaterials in Plant Disease Diagnosis and Management (Mujeebur Rahman Khan, Tanveer Fatima Rizvi, Faheem Ahamad)....Pages 19-33
Bio-Engineered Nanomaterials for Plant Growth Promotion and Protection (Naradala Jayarambabu, Kalagadda Venkateswara Rao)....Pages 35-48
Zinc-Based Nanostructures in Plant Protection Applications (Manal Mostafa, Hassan Almoammar, Kamel A. Abd-Elsalam)....Pages 49-83
Botrytis Gray Mold Nano- or Biocontrol: Present Status and Future Prospects (Esraa Gabal, Amal-Asran, Mohamed A. Mohamed, Kamel A. Abd-Elsalam)....Pages 85-118
Bionanoparticles as Antimicrobial Agents (Karabi Biswas, Sankar Narayan Sinha)....Pages 119-127
Nanopesticides: Synthesis, Formulation and Application in Agriculture (Priyanka Priyanka, Dileep Kumar, Kusum Yadav, Anurag Yadav)....Pages 129-143
Nanobiopesticides for Crop Protection (P. S. Vimala Devi, P. Duraimurugan, K. S. V. P. Chandrika, B. Gayatri, R. D. Prasad)....Pages 145-168
Nano-biopesticides: Synthesis and Applications in Plant Safety (Bipin D. Lade, Dayanand P. Gogle)....Pages 169-189
Nanoscale Fertilizers: Harnessing Boons for Enhanced Nutrient Use Efficiency and Crop Productivity (Anu Kalia, Sat Pal Sharma, Harleen Kaur)....Pages 191-208
Nanodiagnostic Techniques in Plant Pathology (Tahsin Shoala)....Pages 209-222
Role of Nanotechnology Applications in Plant-Parasitic Nematode Control (Al-Kazafy Hassan Sabry)....Pages 223-240
Cytotoxic Potential of Plant Nanoparticles (Ahmed A. Haleem Khan)....Pages 241-265
Engineered Nanoparticle-Based Approaches to the Protection of Plants Against Pathogenic Microorganisms (Nariman Maroufpoor, Mehrdad Alizadeh, Hamed Hamishehkar, Behnam Asgari Lajayer, Mehrnaz Hatami)....Pages 267-283
Back Matter ....Pages 285-291

Citation preview

Nanotechnology in the Life Sciences

Kamel A. Abd-Elsalam Ram Prasad Editors

Nanobiotechnology Applications in Plant Protection Volume 2

Nanotechnology in the Life Sciences Series Editor Ram Prasad School of Environmental Science and Engineering Sun Yat-sen University, Guangzhou, China Amity Institute of Microbial Technology Amity University, Noida, Uttar Pradesh, India

Nano and biotechnology are two of the 21st century’s most promising technologies. Nanotechnology is demarcated as the design, development, and application of materials and devices whose least functional make up is on a nanometer scale (1 to 100 nm). Meanwhile, biotechnology deals with metabolic and other physiological developments of biological subjects including microorganisms. These microbial processes have opened up new opportunities to explore novel applications, for example, the biosynthesis of metal nanomaterials, with the implication that these two technologies (i.e., thus nanobiotechnology) can play a vital role in developing and executing many valuable tools in the study of life. Nanotechnology is very diverse, ranging from extensions of conventional device physics to completely new approaches based upon molecular self-assembly, from developing new materials with dimensions on the nanoscale, to investigating whether we can directly control matters on/in the atomic scale level. This idea entails its application to diverse fields of science such as plant biology, organic chemistry, agriculture, the food industry, and more. Nanobiotechnology offers a wide range of uses in medicine, agriculture, and the environment. Many diseases that do not have cures today may be cured by nanotechnology in the future. Use of nanotechnology in medical therapeutics needs adequate evaluation of its risk and safety factors. Scientists who are against the use of nanotechnology also agree that advancement in nanotechnology should continue because this field promises great benefits, but testing should be carried out to ensure its safety in people. It is possible that nanomedicine in the future will play a crucial role in the treatment of human and plant diseases, and also in the enhancement of normal human physiology and plant systems, respectively. If everything proceeds as expected, nanobiotechnology will, one day, become an inevitable part of our everyday life and will help save many lives. More information about this series at http://www.springer.com/series/15921

Kamel A. Abd-Elsalam  •  Ram Prasad Editors

Nanobiotechnology Applications in Plant Protection Volume 2

Editors Kamel A. Abd-Elsalam Agricultural Research Center Plant Pathology Research Institute Giza, Egypt

Ram Prasad School of Environmental Science and Engineering Sun Yat-sen University Guangzhou, China Amity Institute of Microbial Technology Amity University Noida, Uttar Pradesh, India

ISSN 2523-8027     ISSN 2523-8035 (electronic) Nanotechnology in the Life Sciences ISBN 978-3-030-13295-8    ISBN 978-3-030-13296-5 (eBook) https://doi.org/10.1007/978-3-030-13296-5 © Springer Nature Switzerland AG 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

Plant diseases are caused by bacteria, fungi, insects, nematodes, phytoplasmas, and viruses; the diseases provoked by these pests cause financial losses by reducing attainable yields, product quality, and/or shelf life. Only in the United States, over $600 million is expended annually on fungicides in challenge to control plant pathogens. Traditional plant protection strategies often prove insufficient, considering also that the application of chemical‐based pesticides has negative effects on animals and human beings apart from causing decline in soil fertility. Recent industrial advancements have led to the fabrication of nanomaterials of diverse sizes and shapes. These innovations are the base for further engineering to create unique properties targeted toward specific applications. Nanotechnology would deliver green and efficient alternatives for the management of plant diseases without harming the environment, while the most favorable strategies, in recent scenario, are the use of micro‐ and nanotechnology to promote a more efficient assembly and then release of specific and environmentally sustainable active principles. The wide range of nanotechnology applications in agriculture also includes nanopesticides for the control of plant pathogen interactions and provides new techniques for crop disease control. However, its use in agriculture, especially for plant protection and production, is an under-explored area in the research community. Nanotechnology has many applications in all stages of production, processing, storing, packaging, and transport of agricultural products. Moreover, it will revolutionize the agriculture and food industry by using new and innovative techniques such as precision farming techniques; enhancing the ability of plants to absorb nutrients; improving seed germination and growth, more efficient and targeted use of inputs, plant protection, and pathogen and pesticide/herbicide residue detection; controlling diseases; withstanding environmental pressures; and creating effective systems for processing, storage, and packaging. This book deals with the application of nanotechnology for quicker, more cost-­effective, and precise diagnostic procedures of plant diseases. Additionally, the combination of nanotechnology with microfluidic systems has been effectively applied in molecular plant pathology and can be adapted to detect specific

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pathogens and toxins. Moreover, the application of nanotechnology in plant disease ­control, antimicrobial mechanisms, and nanotoxicity on plant ecosystem is ­discussed in detail. The second volume of the book focuses on additional information on the applications of nanotechnology in plant protection and plant pathology. In Chap. 1, Ganguli reviews the intellectual property rights aspects of nano-­ biopesticides. In Chap. 2, Khan et al. highlight the application of nanomaterials in plant disease diagnosis and management. In Chap. 3, Jayarambabu et al. describe bio-engineered nanomaterials for plant growth promotion and protection. In Chap. 4, Mostafa et al. describe zinc-based nanostructures in plant protection applications. In Chap. 5, Gabal et al. focus on Botrytis gray mold nano- or biocontrol: present status and future prospects. In Chap. 6, Biswas and Sinha highlight on the bio-­ nanoparticles as antimicrobial agents. In Chap. 7, Priyanka et al. discuss nanopesticides: synthesis, formulation, and application in agriculture. In Chap. 8, Vimala Devi et al. focus on nano-biopesticides for crop protection. In Chap. 9, Lade et al. examine nano-biopesticide: synthesis and applications in plant safety. In Chap. 10, Kalia details on nanoscale fertilizers: harnessing boons for enhanced nutrient use efficiency and crop productivity. In Chap. 11, Shoala et  al. give an overview of nanodiagnostic techniques in phytopathogens. In Chap. 12, Hassan Sabry discusses the role of nanotechnology applications in plant parasitic nematode control. In Chap. 13, Haleem Khan explains the cytotoxic potential of plant. Finally, Chap. 14 highlights the applications of engineered nanomaterials against plant pathogenic microorganisms. We wish to thank the Springer officials, particularly William F.  Curtis, Eric Schmitt, Eric Stannard, and Rahul Sharma, for their generous support and efforts in accomplishing this volume. We are highly delighted and thankful to all our contributing authors for their vigorous support and outstanding cooperation to write altruistically these authoritative and valuable chapters. We specially thank our families for their consistent support and encouragement. With a bouquet of information on the different aspects of plant protections from nanomaterials, the editors hope that this book is a valuable resource for the students of different divisions; the researchers and academicians, working in the field of nanoscience, nanotechnology, plant sciences, agriculture microbiology, and fungal biology; and the scholars interested in strengthening their knowledge in the area of nanobiotechnology. Giza, Egypt Noida, Uttar Pradesh, India 

Kamel A. Abd-Elsalam Ram Prasad

Contents

  1 Intellectual Property Rights in Nano-biopesticides������������������������������    1 Prabuddha Ganguli   2 Application of Nanomaterials in Plant Disease Diagnosis and Management ��������������������������������������������������������������������������������������������   19 Mujeebur Rahman Khan, Tanveer Fatima Rizvi, and Faheem Ahamad   3 Bio-Engineered Nanomaterials for Plant Growth Promotion and Protection������������������������������������������������������������������������������������������   35 Naradala Jayarambabu and Kalagadda Venkateswara Rao   4 Zinc-Based Nanostructures in Plant Protection Applications ������������   49 Manal Mostafa, Hassan Almoammar, and Kamel A. Abd-Elsalam   5 Botrytis Gray Mold Nano- or Biocontrol: Present Status and Future Prospects ������������������������������������������������������������������������������   85 Esraa Gabal, Amal-Asran, Mohamed A. Mohamed, and Kamel A. Abd-Elsalam   6 Bionanoparticles as Antimicrobial Agents��������������������������������������������  119 Karabi Biswas and Sankar Narayan Sinha   7 Nanopesticides: Synthesis, Formulation and Application in Agriculture�������������������������������������������������������������������������������������������  129 Priyanka Priyanka, Dileep Kumar, Kusum Yadav, and Anurag Yadav   8 Nanobiopesticides for Crop Protection��������������������������������������������������  145 P. S. Vimala Devi, P. Duraimurugan, K. S. V. P. Chandrika, B. Gayatri, and R. D. Prasad   9 Nano-biopesticides: Synthesis and Applications in Plant Safety ��������  169 Bipin D. Lade and Dayanand P. Gogle

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10 Nanoscale Fertilizers: Harnessing Boons for Enhanced Nutrient Use Efficiency and Crop Productivity������������������������������������  191 Anu Kalia, Sat Pal Sharma, and Harleen Kaur 11 Nanodiagnostic Techniques in Plant Pathology������������������������������������  209 Tahsin Shoala 12 Role of Nanotechnology Applications in Plant-Parasitic Nematode Control������������������������������������������������������������������������������������  223 Al-Kazafy Hassan Sabry 13 Cytotoxic Potential of Plant Nanoparticles��������������������������������������������  241 Ahmed A. Haleem Khan 14 Engineered Nanoparticle-Based Approaches to the Protection of Plants Against Pathogenic Microorganisms��������������������������������������  267 Nariman Maroufpoor, Mehrdad Alizadeh, Hamed Hamishehkar, Behnam Asgari Lajayer, and Mehrnaz Hatami Index������������������������������������������������������������������������������������������������������������������  285

Contributors

Kamel A. Abd-Elsalam  Plant Pathology Research Institute, Agricultural Research Center (ARC), Giza, Egypt Unit of Excellence in Nano-Molecular Plant Pathology Research – Plant Pathology Research Institute, Giza, Egypt Faheem  Ahamad  Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh, India Mehrdad  Alizadeh   Department of Plant Pathology, College of Agriculture, Tarbiat Modares University, Tehran, Iran Hassan  Almoammar  ETH Zürich, Department of Biology, Institute of Microbiology, Zürich, Switzerland Amal-Asran  Plant Pathology Research Institute, Agricultural Research Center (ARC), Giza, Egypt Unit of Excellence in Nano-Molecular Plant Pathology Research Center  – Plant Pathology Research Institute, Giza, Egypt Karabi Biswas  Environmental Microbiology Research Laboratory, Department of Botany, University of Kalyani, Kalyani, West Bengal, India K. S. V. P. Chandrika  ICAR-Indian Institute of Oilseeds Research, Hyderabad, Telangana, India P.  Duraimurugan  ICAR-Indian Institute of Oilseeds Research, Hyderabad, Telangana, India Esraa Gabal  Agricultural Science and Resource Management in the Tropics and Subtropics, Faculty of Agriculture, Bonn University, Bonn, Germany Prabuddha Ganguli  Rajiv Gandhi School of Intellectual Property Rights, Indian Institute of Technology, Kharagpur, West Bengal, India

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B.  Gayatri  ICAR-Indian Institute of Oilseeds Research, Hyderabad, Telangana, India Dayanand  P.  Gogle  Department of Molecular Biology & Genetic Engineering, RTM Nagpur University, Maharashtra, India Ahmed A. Haleem Khan  Department of Botany, Dichpally, Telangana University, Nizamabad, Telangana, India Hamed  Hamishehkar  Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Mehrnaz  Hatami  Department of Medicinal Plants, Faculty of Agriculture and Natural Resources, Arak University, Arak, Iran Institute of Nanoscience and Nanotechnology, Arak University, Arak, Iran Naradala Jayarambabu  Centre for Nano Science and Technology, IST, Jawaharlal Nehru Technological University, Hyderabad, Telangana, India Anu Kalia  Electron Microscopy and Nanoscience Laboratory, Department of Soil Science, College of Agriculture, Punjab Agricultural University, Ludhiana, Punjab, India Harleen  Kaur  Department of Microbiology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana, Punjab, India Mujeebur Rahman Khan  Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh, India Dileep  Kumar  Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India Bipin D. Lade  Department of Molecular Biology & Genetic Engineering, RTM Nagpur University, Maharashtra, India Diksha  B.  Lade  DNA Fingerprinting Lab, Seed Testing Laboratory, Nagpur, Maharashtra, India Behnam  Asgari  Lajayer  Department of Soil Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran Nariman  Maroufpoor   Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran Mohamed A. Mohamed  Plant Pathology Research Institute, Agricultural Research Center (ARC), Giza, Egypt Manal  Mostafa  CIHEAM IAMB  – Mediterranean Agronomic Institute of Bari, Valenzano, Bari, Italy Plant Pathology Research Institute, Agricultural Research Center (ARC), Giza, Egypt

Contributors

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R. D. Prasad  ICAR-Indian Institute of Oilseeds Research, Hyderabad, Telangana, India Ram  Prasad  School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou, China Amity Institute of Microbial Technology, Amity University, Noida, Uttar Pradesh, India Priyanka  Priyanka  Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India Kalagadda  Venkateswara  Rao  Centre for Nano Science and Technology, IST, Jawaharlal Nehru Technological University, Hyderabad, Telangana, India Tanveer  Fatima  Rizvi  Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh, India Al-Kazafy  Hassan  Sabry  Pests and Plant Protection Department, National Research Centre, Cairo, Egypt Sat Pal Sharma  Department of Vegetable Science, College of Agriculture, Punjab Agricultural University, Ludhiana, Punjab, India Tahsin  Shoala  Department of Environmental Biotechnology, College of Biotechnology, Misr University for Science and Technology, Giza, Egypt Sankar  Narayan  Sinha  Environmental Microbiology Research Laboratory, Department of Botany, University of Kalyani, Kalyani, West Bengal, India P.  S.  Vimala  Devi  ICAR-Indian Institute of Oilseeds Research, Hyderabad, Telangana, India Anurag  Yadav  College of Basic Sciences & Humanities, Sardarkrushinagar Dantiwada Agricultural University, S.K. Nagar, Banaskantha, Gujarat, India Kusum  Yadav  Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh, India

About the Editors

Kamel  A.  Abd-Elsalam  Ph.D., is a head researcher at Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt. His research interests include developing, improving, and deploying plant biosecurity diagnostic tools, understanding and exploiting fungal pathogen genomes, and developing eco-friendly hybrid nanomaterials for controlling toxicogenic fungi and plant diseases. He has published 8 book chapters, 6 review articles, 1 translated book, and more than 120 research articles in international peerreviewed journals, including Fungal Diversity, Fungal Biology, FEMS Microbiology Ecology, PLOS One, and PLOS Genetics. He is an associate editor for Mycosphere, a review editor for Frontiers in Genomic Assay Technology, and a referee for journals, including the Plant Pathology, Journal of Phytopathology, Crop Protection, IET Nanotechnology, Fungal Diversity, BMC Genomics, and Foodborne Pathogens and Disease. He also edited or authored some books. He has also served as molecular mycologist for 5 years in the Department Botany and Microbiology, College of Science, King Saud University, Saudi Arabia. He received the Federation of Arab Scientific Research Councils Prize for Distinguished Scientific Research in Biotechnology (fungal genomics) in 2014 (first ranking). He has pursued his Ph.D. in Molecular Plant Pathology from Christian Albrechts University of Kiel (Germany), where he has been awarded Postdoctoral Fellowship in 2008, and Suez Canal University (Egypt). Moreover, he served as a visiting associate xiii

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professor in the Institute of Excellence in Fungal Research, Mae Fah Luang University, Thailand; Institute of Microbiology, TUM, Germany; Laboratory of Phytopathology, Wageningen University, the Netherlands; and Plant Protection Department, Sassari University, Italy. Ram Prasad  Ph.D., is assistant professor at the Amity Institute of Microbial Technology, Amity University, Uttar Pradesh, India. His research interest includes plant-microbe interactions, sustainable agriculture, and microbial nanobiotechnology. He has more than a hundred publications to his credit, including research papers and book chapters, five patents issued or pending, and edited or authored several books. He has 11 years of teaching experience and has been awarded the Young Scientist Award (2007) and Prof. J.  S. Datta Munshi Gold Medal (2009) by the International Society for Ecological Communications, the FSAB Fellowship (2010) by the Society for Applied Biotechnology, the Outstanding Scientist Award (2015) in the field of microbiology by Venus International Foundation, and the American Cancer Society UICC International Fellowship for Beginning Investigators (USA, 2014). In 2014–2015, he served as a visiting assistant professor in the Department of Mechanical Engineering at Johns Hopkins University, USA.

Chapter 1

Intellectual Property Rights in Nano-biopesticides Prabuddha Ganguli

Contents 1.1  Nano-biopesticides—An Overview 1.2  Intellectual Property Rights—Where Does IPR Meet Nano-biopesticides 1.3  Introduction to IPR Tools (WIPO and WTO) 1.4  Patenting Trends in Nano-biopesticides 1.5  Mitigating Potential Risks 1.6  Conclusion References

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1.1  Nano-biopesticides—An Overview The worldwide biopesticide market, which are biological pesticides derived from natural materials such as plants, animals and bacteria [comprising bioinsecticides, biofungicides, bioherbicides, bionematicides, rodenticides, minerals and plant incorporated protectants (PIP)], is projected to grow at a CAGR of 13.9% till 2025 and reach a value of US$ 9.5bn by the end of 2025 (Transparency Market Research 2017). The key features driving the growth of “Green Agriculture” and “Integrated Pest Management” (IPM) programmes are the sustained need for controlled use of pesticides and fertilizers through environmentally friendly approaches for enhanced efficacy with simultaneous reduction of the amount of active ingredients (AIs) through targeted delivery of the AIs to specific sites, minimised negative environmental impact and preventing infusion of toxic residues in the agro-food value chain (Kumar et al. 2019).

P. Ganguli (*) Rajiv Gandhi School of Intellectual Property Rights, Indian Institute of Technology, Kharagpur, West Bengal, India © Springer Nature Switzerland AG 2019 K. A. Abd-Elsalam, R. Prasad (eds.), Nanobiotechnology Applications in Plant Protection, Nanotechnology in the Life Sciences, https://doi.org/10.1007/978-3-030-13296-5_1

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The global biopesticides market is fragmented in nature, though recent trends in consolidation of positions are led by strategic mergers and acquisitions amongst big players. Start-ups and small innovative companies have sprouted in fairly large numbers in the biopesticides R&D space especially related to their development and application. Interestingly, the field of nano-biopesticides as “smart delivery systems of biopesticides” is still in its very early days, aggressively exploring commercial feasibilities. Conceptual breakthroughs have been reported with regard to their controlled release kinetics, enhanced permeability, stability, solubility, prevention of premature degradation of active ingredients (AIs) under harsh environment ­conditions, pesticide loss due to leaching and evaporation, functionality as biosensors for detection of pathogens and pesticide residues, etc. Scientific published literature in the last two decades is replete with promising results in relatively safer application of biopesticides in pre-harvest and post-harvest agricultural practices as compared to chemical pesticides (Nuruzzaman et al. 2016). Advances in nano-technology leading to the creation of new processes and novel materials with surprising properties opened up wide ranging opportunities in their applications in precision agriculture and food technologies (Duhan et  al. 2017; Athanassiou et al. 2018). The active intervention of “Environmental Regulatory Agencies”, “Legislations including the strategic management of Intellectual Property Rights” and “Ethical Overtones” in various jurisdictions needs to be taken into consideration to ensure balanced innovation, commercialisation and fair distribution of value in the marketplace. In recent times, national competition authorities have also begun to play a significant role in regulating the global agro-market dynamics by closely examining IPR licencing deals, collaborations, mergers and acquisitions involving diverse stakeholders. The interface of nanotechnology [materials measuring between ~1  nm and ~100 nm] as applied in the field of biopesticides and Intellectual Property Rights is to be viewed from these interlaced and/or intercalated perspectives.

1.2  I ntellectual Property Rights—Where Does IPR Meet Nano-biopesticides Intellectual Property Rights (IPR) offers a workable legal framework for ownership in the knowledge space thereby ensuring due recognition and reasonable benefits to the creator/owner of that knowledge and offering him protection and incentive to share his knowledge with the society via fair “knowledge prospecting”. Further, traditional knowledge in the public domain that has evolved over generations is recognised as “prior art”, and in certain jurisdictions when utilised for development of new commercially exploitable knowledge, must involve fair sharing of benefits between the community liked to the relevant traditional knowledge and the present

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innovator utilising the traditional knowledge. It is also expected that the innovation process ought to prosper without damage to the environment including plant, animal and human life, and therefore it would be prudent to have such and other ethical & public interest issues incorporated as limitations in the intellectual property laws. It is hoped that a balanced and ethical framework of social governance and knowledge management would encourage development of innovations and advance a sense of respect for owned knowledge, discourage “knowledge piracy”, “free riding”, “profiteering from counterfeits”, and “overuse/misuse” of IPR, thereby founding symbiotic relationships for the positive development of a free and fair society. However, for effective and ethical functioning of a strong and just IPR system, balancing legislations are required to simultaneously encourage innovations and discourage misuse of IPR and unfair/anti-social monopolistic practices. These considerations are most relevant in the context of IPR when applied to knowledge dynamics in the preharvest and post-harvest agricultural practises. The WTO’s Agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPS), negotiated during the 1986–94 Uruguay Round, introduced intellectual property rules into the multilateral trading system for the first time [WTO]. The TRIPS Agreement covers five broad areas, namely, (i) how general provisions and basic principles of the multilateral trading system apply to international intellectual property, (ii) what the minimum standards of protection are for intellectual property rights that members should provide, (iii) which procedures members should provide for the enforcement of those rights in their own territories, (iv) how to settle disputes on intellectual property between members of the WTO, and (v) special transitional arrangements for the implementation of TRIPS provisions. Whilst the WTO agreements entered into force on 1 January 1995, the TRIPS Agreement allowed WTO members certain transition periods before they were obliged to apply all of its provisions. Developed country members were given 1 year to ensure that their laws and practices conform to the TRIPS Agreement. Developing country members and (under certain conditions) transition economies were given 5 years, until 2000. Least-developed countries initially had 11 years, until 2006— now extended to 1 July 2021 in general. In November 2015, the TRIPS Council agreed to further extend exemptions on pharmaceutical patent and undisclosed information protection for least-developed countries until 1 January 2033 or until such date when they cease to be a least-­ developed country member, whichever date is earlier. They are also exempted from the otherwise applicable obligations to accept the filing of patent applications and to grant exclusive marketing rights during the transition period. The areas of intellectual property are covered by Trade Related Aspects of Intellectual Property Rights (TRIPS) Agreement under the WTO are Copyright and Related Rights, Trademarks, Geographical Indications, Industrial Designs, Patents, Layout-Designs (Topographies) of Integrated Circuits, Protection of Undisclosed Information and Control of Anti-Competitive Practices in Contractual Licences.

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In the context of innovations in the field of nano-biopesticides all the fields of IPR listed in the TRIPs Agreement with the exception of Layout-Designs (Topographies) of Integrated Circuits are relevant.

1.3  Introduction to IPR Tools (WIPO and WTO)

IPR tool Patent

What it protects A patent is an exclusive right granted by the government for an invention that satisfies criteria set for patentable subject matter, and further is new, involves an inventive step and is capable of industrial application. It gives its owner the legal right within the territory in which the patent is granted to exclude or stop others from making, using, offering for sale, selling or importing a product or process based on the patented invention. In many countries, some types of incremental inventions or small adaptations of existing products are protectable as utility models.

Utility models (also known as “short-­ term patents,” “petty patents” or “innovation patents”) Industrial designs Exclusivity over the ornamental or aesthetic features of a product can be protected through laws on industrial designs, in some countries referred to as “design patents”. Trademarks Trademark protection provides exclusivity over words, marks and colours used to distinguish the products of one company from those of another. Copyright and The form of expression of original literary, artistic and technical works related rights (such as software) may be protected by copyright and related rights. Geographical A name or indication associated with a place is sometimes used to indications identify a product. This “geographical indication” does not only say where the product comes from. More importantly, it identifies the product’s special characteristics, which are the result of the product’s origins. Using the indication when the product was made elsewhere or when it does not have the usual characteristics can mislead consumers, and can lead to unfair competition. The TRIPS Agreement says members have to provide ways to prevent such misuse of geographical indications. For wines and spirits, the TRIPS Agreement provides higher levels of protection, i.e. even where there is no danger of the public being misled. Some exceptions are allowed, for example if the term in question is already protected as a trademark or if it has become a generic term. In many countries, a breeder of a new plant variety may obtain protection Protection of new in the form of “plant breeder’s rights.” plant varieties of plants Trade secrets All types of confidential business information, including secret designs, machines and processes, may be protected as trade secrets so long as the information is not generally known, its commercial value derives from its secrecy, and reasonable steps have been taken to keep it secret (for example, restricting access on a “need to know” basis, and entering into confidentiality or non-disclosure agreements. (continued)

1  Intellectual Property Rights in Nano-biopesticides IPR tool Layout-design (or topography) of integrated circuits. Anti-competitive practices in contractual licences

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What it protects An original layout-design of an integrated circuit may be protected against copying. One way for a right holder to commercially exploit his or her intellectual property rights includes issuing a licence to someone else to use the rights. Recognising the possibility that right holders might include conditions that are anti-competitive, the TRIPS Agreement says that under certain conditions, governments have the right to take action to prevent anti-competitive licencing practices. It also says governments must be prepared to consult each other on controlling anti-competitive licencing practices.

As the field of nano-biopesticides is largely invention-led, patent is the most relevant IPR tool that is globally exploited. Hence the a few essential aspects of patents are being elaborated in the next few sections of this chapter. It ought to be noted that trademarks play a significant role in the naming of the products and services. Copyright and design registrations are applicable for the protection of distinctive packaging, instruction booklets, advertising, etc. A patent is an exclusive right granted by the government for an invention that satisfies criteria set for patentable subject matter, and further is new, involves an inventive step and is capable of industrial application. It gives its owner the legal right to exclude or stop others from making, using, offering for sale, selling or importing a product or process based on the patented invention. A patent is granted by the national patent office of a country or a regional patent office for a group of countries. It is valid for a limited period of time, generally 20 years from the date of filing the application, provided the required maintenance fees are paid on time. A patent is a territorial right, limited to the geographical boundary of the relevant country or region in which it is granted. In return for the exclusive right provided by a patent, the applicant is required to disclose the invention to the public by providing a detailed, accurate and complete written description of the invention in the patent application. The granted patent and, in many countries, the patent application are published in an official journal or gazette. Further, the International Patent Classification System (IPC) provides hierarchical classification system used to classify and search patent documents. It also serves as an instrument for the orderly arrangement of patent documents, a basis for selective dissemination of information and for investigating the state of the art in given fields of technology. The IPC consists of eight sections, which are divided into 120 classes, 628 subclasses and approximately 70,000 groups. The eight sections are Human Necessities; Performing Operations; Transporting; Chemistry; Metallurgy; Textiles; Paper; Fixed Constructions; Mechanical Engineering; Lighting; Heating; Weapons; Blasting; Physics and Electricity. Developers of nano-biopesticides are therefore engaged in creating strategic portfolio of patents in countries of their business interests, further monetizing them through their commercialised products in the markets and/or transacting the patents through licencing, assignments, cross-licencing, etc.

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Further, patent information is available in the public domain as a rich structured source of technical and legal information on inventions done in various parts of the world. Patent information and its judicious use promote further inventions and the creation/running of sustained businesses.

1.4  Patenting Trends in Nano-biopesticides A publication in 2015 listing the publications in patent and non-patent literature related to nanomaterials in plant protection and fertilisers (Gogos et  al. 2012) reported that most patents in these fields were held by companies like BASF, Dow Agrosciences, Evonik-Degussa, Monsanto, Rhone-Poulec and other relatively smaller companies. This article also reported a list of patents that claimed pesticide products with particles mostly in the nanosize ranges, wherein the function of the nanomaterial varied from being an additive (dispersing agent, UV protection agent, carries, active ingredient, bio-delivery agent, controlled release). Commercially available products reported were Primo MAXX, Banner (as nano emulsions from Syngenta), Karate ZEON (Capsules with λ-cyhalotrin from Sygenta), Demand CS Capsules with pyrethroid (λ-cyhalotrin from Sygenta), ECOFLEX (Aliphatic copolyester, “nanofibre” as a pheromone dispenser from BASF), Aerosil 200 (SiO2 from Sygenta), Trico TiO2 23.2% Sheepgrease (pesticide from Omya AG), FEROX Zero-valent iron nanopowder (soil remediation from ARS Technologies, Inc) and SoilSet SiO2 (soil management from Sequoia Pacific Research Company). Other commercial products reported are CLARIVA® (from Syngenta International AG, for seed treatment based on natural soil bacteria to protect a plant’s root from nematodes, especially for soybeans), SIVANTO® prime (from Bayer AG which is a nature-derived insecticide for efficient control of key adult and immature sucking pests, such as Aphids, whiteflies and psyllids, scales, leaf miners, beetles mirids and selected hoppers, for vegetables, fruits as well as cotton and soybeans), Nimbecidine EC with 0.03% Azadirachtin (from T. Stanes & Company Ltd., India) for multiple modes of action like anti-feedant, repellent, ovi-position deterrent, insect growth regulator for whiteflies, thrips, aphids, caterpillars, mealy bugs, and leafhoppers, and sterilant), Nano-Gro™ (from Agro Nanotechnology Corp. Plant growth regulator and immunity enhancer), NanoMax-NPK (from JU Agri Sciences, India for promoting growth of green leaves, photosynthesis, carbohydrates, oil fats and proteins in crops), Nano-Ag Answer® (from Urth Agriculture, for promoting high yield along with a reduction in watering [~20%] and predatory pests) and Uremic Nano Fertilizer offering plant nutrition with high uptake efficiency and avoiding urea degradation in the presence of sun and heat and lowering damage to soil structure due to the effects of urea (Kumar et al. 2019). Nano-5® from Uno Fortune Inc is a 3-in-1 Natural Mucilage Organic Fertilizer which promotes cleavage of plant cells, differentiation, proliferation, blooming, fruiting, resistance to cold, drought and ethene, prevention of fruit dropping and

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splitting, adjusting and shortening the period of production. It further provides prevention and cure of diseases and pests by preventing fusarium, fungi, bacterial wilt, bacteria, virus and pests inhibiting piercing-sucking pests, chewing pests, nematodes, and seasonal diseases and pests (Uno Fortune). A recent review of covering antimicrobial agents (e.g. silver, titanium dioxide), nano-bio pesticides (hydrophobic silica) and smart delivery systems (polymeric nanoparticles) also listed patents claiming pesticides, anionic herbicides, fertilisers, fungicides, agrochemicals, insect repellants and essential oils in diverse carrier materials such as clays, silicas and polymers (Narayanan et al. 2013). This article further reviewed the development of nano-biosensors for diagnosis of pests and pesticide residues including the use of quantum dots applying the Forster Resonance Energy Transfer (FRET) technology, polymeric particles, clay clays, silica particles, magnetic nanoparticles, nanotitanium dioxides, nanoemulsions, nanocomposites, nanofibers, nanotubes, etc. Patent Analytics Report covering agricultural nanomaterial released in January 2016 by IP Australia (IP Australia 2016) reviewed the patent status of nanomaterials in agriculture. With specific reference to protection of plants, the technology categories (patent families) covered are bioavailability/activity enhancement, stability enhancement, agent per se, inert carrier, sustained/delayed pesticide release and plant protection. BASF, Shikefeng Chemical Co Ltd., Suzhou Setek Co Ltd., Vive Crop Protection Inc, Dow Agrosciences LLC were reported as the key patent players in these technology categories. Relecura Technologies Pvt. Ltd, Bangalore, India [Relecura] is a specialised group in patent search and technology mapping. At the request of the author of this chapter, Dr. Murari Venkataraman and Dr. George Koomullil of Relecura conducted a specialised search of patenting activity involving nano-biopesticides. The results of Relecura’s search were analysed, and the raw data are reproduced in this chapter with permission of Relecura. Patenting inventions in nano-biopesticides since 2001 is depicted in Fig.  1.1. Filing of patents in nano-biopesticides is a fairly recent activity which has picked up since 2006 from just 16 filings to about 300 filings in 2017. Clearly, developments in basic science of biopesticides and their allied aspects also accelerated during the same period with patent filings in biopesticides increasing from about 1200 in 2006 to about 11,000 in 2018. Figure 1.2 presents a snapshot of the geographical spread and intensity of patent filings in nano-biopesticides. China has become a lead country for patent applications followed by US, Europe, India, Canada, Japan, Korea, Australia and others. It is to be noted that WO designates the patent publications of the Patent Cooperation Treaty Application (administered by WIPO). As patenting transcends beyond academic borders into the commercial world, it is pertinent to explore the origins of the inventing organisations/people in the field of nano-biopesticides. Table  1.1 presents the top patent application holders. DowDupont leads the pack, followed by Vive Crop Protection INC, BASF, ICEUTICA, JIANGSU, BAYER, BIONANOPLUS and others.

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217

2018

139

222

2016

298

166

2017

2015

2014

2013

2012

2011

2004

2010

2003

2009

2002

7 16 24 34 24 31 46 45 2008

5

2007

6

2006

2

2005

2 2001

83

Fig. 1.1  Nano-biopesticide patent applications 700

652

600 500 400 300

214 144

200 100

92

76

47

43

17

17

JP

KR

AU

WO

73

0 CN

US

EP

IN

CA

Others

Fig. 1.2  Nano-biopesticide patent applications in various countries

The patent applicants listed in Table 1.1, based on their business interests, have filed their patent applications in various jurisdictions which is reported in Table 1.2. DOWDUPONT, having international operations have filed its patent applications in China, USA, European Patent Office (EP) Canada, India and Japan, Korea, Australia Taiwan and Spain. VIVE CROP PROTECTION INC with VIVE CROP PROT INC have filed patent applications in China, USA, European Patent Office (EP) Canada, India and Japan, Korea, Australia and Spain. BASF has filed its patent applications in China, USA, European Patent Office (EP), Canada, India, Japan, Korea and Spain, whereas ICEUTICA PTY LTD has filed its patent applications in China, USA, European Patent Office (EP), Canada, India, Korea, Australia and Spain. The Chinese applicants, except for CHEMCHINA, have concentrated only in China. The Japanese companies have concentrated their applications in Japan. Companies such as BIONANOPLUS S L, DENDRITIC NANOTECHNOLOGIES INC, AGFORM LIMITED, INSTILLO GMBH, RAG-STIFTUNG, 3M, A I INNOVATIONS N V have made inroads with specialised technologies, whereas

1  Intellectual Property Rights in Nano-biopesticides Table 1.1  Top patent applicants in nano-biopesticides

Top patent applicants DOWDUPONT VIVE CROP PROTECTION INC BASF ICEUTICA PTY LTD JIANGSU ROTAM CHEMISTRY BAYER BIONANOPLUS S L MAANSHAN KEBANG ECO FERTILIZER NAGAURA YOSHIAKI SHANDONG SUNWAY LANDSCAPE TECHNOLOGY VIVE CROP PROT INC CHINESE ACADEMY OF SCIENCES CHEMCHINA DENDRITIC NANOTECHNOLOGIES INC AGFORM LIMITED INSTILLO GMBH RAG-STIFTUNG UNIVERSITY OF CALIFORNIA 3M A I INNOVATIONS N V

9 Number of applications 176 25 23 18 17 14 13 12 11 11 10 9 8 8 7 7 7 7 6 6

The University of California’s Center for Environmental Implications of Nanotechnology (UC CEIN) is working to ensure the responsible use and safe implementation of nanotechnology in the environment through a multi-disciplinary approach to research, knowledge acquisition, education and outreach. As mentioned earlier in the chapter, the International Patent Classification System (IPC) provides a hierarchical classification system used to classify and search patent documents. The Cooperative Patent Classification (CPC) system, in force from 1 January 2013, is a bilateral system which has been jointly developed by the EPO and the USPTO. Each classification with its subclassification identifies the subject matter that is addressed in the invention. Table 1.3 lists the most frequently occurring CPC Codes whilst searching nano-biopesticide patents/patent applications. A perusal of Table 1.3 drives home the point that inventions in the field of biopesticides involve the use of heterocyclic compounds of various types; cyclopropane carboxylic acids or derivatives; nitriles, thiocompounds; mixtures of one or more fertilisers with materials not having a specifically fertilising activity but with pesticididal activities; microcapsules; biopesticide dispersions or gels; biopesticides containing material from algae, lichens, bryophyta, multicellular fungi or plants or extracts thereof; biopesticides containing solids as carriers or diluents; macromolecular compounds; biopesticides in shaped forms, e.g. sheets, not provided for in

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Table 1.2  Patent filings in various countries by the patent applicants in nano-biopesticides Patent applicants DOWDUPONT VIVE CROP PROTECTION INC BASF ICEUTICA PTY LTD JIANGSU ROTAM CHEMISTRY BAYER BIONANOPLUS S L MAANSHAN KEBANG ECO FERTILIZER NAGAURA YOSHIAKI SHANDONG SUNWAY LANDSCAPE TECHNOLOGY VIVE CROP PROT INC CHINESE ACADEMY OF SCIENCES CHEMCHINA DENDRITIC NANOTECHNOLOGIES INC AGFORM LIMITED INSTILLO GMBH RAG-STIFTUNG UNIVERSITY OF CALIFORNIA 3M A I INNOVATIONS N V

CN US EP CA IN JP KR AU 25 49 29 23 15 2 12 2 2 9 3 5 1 1 0 2 2 2 7 2 2 3 2 0 4 3 3 3 2 0 1 2 17 0 0 0 0 0 0 0 6 2 1 2 0 0 0 1 1 2 4 2 2 2 0 0 12 0 0 0 0 0 0 0 0 0 0 0 0 11 0 0 11 0 0 0 0 0 0 0 1 9 0 1 1 1 1 2 0 1

0 0 2 1 1 1 2 2 2 2

3 0 1 2 1 1 1 1 4 1

4 0 1 1 0 1 1 2 0 1

0 0 1 1 1 0 1 0 0 1

1 0 1 0 1 1 1 0 0 0

0 0 0 1 0 1 0 0 0 0

1 0 1 0 1 1 0 0 0 0

TW 4 0 0 0 0 1 0 0 0 0

ES 4 0 1 0 0 0 0 0 0 0

0 0 0 1 0 0 0 0 0 0

0 0 0 0 1 0 0 0 0 0

any other group of this main group; applications of nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery. The inventions in the patents/patent applications claim a range of benefits such as targeted delivery at a predetermined site, controlled delivery, enhanced solubility of the active ingredients (if insoluble or sparingly soluble), increased stability, dispersibility, permeability, wettability, decreased droplet size for better spraying and retention on the substrate, reduced microbial degradation/leaching/evaporation/ drainage/runoff/photolysis, choice of appropriate adjuvants for optimal biopesticidal activity, etc. The nano-materials and phases include polymer and lipid-based nanomaterials, mesoporous inorganic substrates such as silicas, clay-based nanomaterials, layered hydroxides, carbon nano-materials such fullerenes, carbon naoparticles, fullerol and single-walled carbon nanotubes/multiwall carbon nanotubes, nanocapsules, nanospheres, nanogels, micelles, liposomes, nanoemulsions, nanosuspensions, solid lipo-nanoparticles, functionality as biosensors for detection of pathogens and pesticide residues, linking up with communications technologies and in some cases with artificial intelligence. Claims have also been made on encapsulated biopesticides containing material from algae, lichens, bryophyta, multicellular fungi or plants or extracts thereof. Research centres around the world are undertaking research to improve techniques to enhance their commercial feasibility.

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Table 1.3  International Patent Classification of patents/patent applications in nano-biopesticides PC code A01N 43/40 A01N 43/653 C05G 3/02 A01N 43/78 A01N 25/04 A01N 47/36 A01N 43/50 A01N 53/00 C05G 3/00 A01N 43/56 A01N 25/28 A01N 43/54 A01N 43/16 A01N 65/00 A01N 43/90

Number of patent applications 106

Explanation of the CPC codes Biopesticides containing six-membered heterocyclic compounds

89

Biopesticides containing 1,2,4-triazoles; hydrogenated 1,2,4-triazoles

84 83

Mixtures of one or more fertilisers with materials not having a specifically fertilising activity but with pesticides Biopesticides containing 1,3-thiazoles; hydrogenated 1,3-thiazoles

82

Biopesticide dispersions or gels

82

Biopesticides containing the group N—CO—N directly attached to at least one heterocyclic ring; thio-analogues thereof Biopesticides containing 3-diazoles; hydrogenated 1,3-diazoles

79 78

72

Biopesticides containing cyclopropane carboxylic acids or derivatives thereof Mixtures of one or more fertilisers with materials not having a specifically fertilising activity Biopesticides with 1,2-diazoles; hydrogenated 1,2-diazoles

71

Biopesticide microcapsules

71

Biopesticides containing 1,3-diazines; hydrogenated 1,3-diazines

67

Biopesticides containing oxygen as the ring hetero atom

65

Biopesticides containing material from algae, lichens, bryophyta, multicellular fungi or plants or extracts thereof Biopesticides containing two or more relevant hetero rings, condensed amongst themselves or with a common carbocyclic ring system Mixtures of one or more fertilisers with materials not having a specifically fertilising activity but with soil conditioners Biopesticides containing five-membered rings with three hetero atoms Biopesticides containing solids as carriers or diluents

78

61

C05G 3/04 A01N 43/82 A01N 25/08 A01N 37/10 A01N 43/84

57

A01N 25/10

50

54 53 53 52

Biopesticides containing aromatic or araliphatic carboxylic acids, or thio-analogues thereof; derivatives thereof Biopesticides containing six-membered rings with one nitrogen atom and either one oxygen atom or one sulfur sulphur atom in positions 1,4 Biopesticides containing macromolecular compounds (continued)

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Table 1.3 (continued) PC code A01N 25/34 A01N 43/80

Number of patent applications 50

A01N 25/30 A01N 43/60 A01N 37/46 A01N 43/36 B82Y 5/00 C07D 401/04

47

Explanation of the CPC codes Biopesticides of shaped forms, e.g. sheets, not provided for in any other group of this main group Biopesticides containing five-membered rings with one nitrogen atom and either one oxygen atom or one sulfur sulphur atom in positions 1,2 Biopesticides containing by surfactants

47

Biopesticides containing 1,4-diazines; hydrogenated 1,4-diazines

45

Biopesticides containing N-acyl derivatives

44

Biopesticides with five-membered rings

44

A01N 37/34

43

Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery Biopesticide containing heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom directly linked by a ring-member-to-ring-member bond Biopesticides containing nitriles

48

44

Developments in the field of nano-biopesticides embracing the latest techniques have also been patented. Example of one such latest technique has been exclusively licenced to TechAccel of Shawnee Mission, KS in 2015, for a biopesticide application developed at Kansas State University on a pesticide which is useful for agricultural applications as well as pest management for the home and garden. This pesticide claimed in US Patent No. 8841272, entitled “Double-Stranded RNA-Based Nanoparticles for Insect Gene Silencing”, claims a nanoparticle useful for RNA interference of a target insect gene which is composed of a biopolymer matrix and an insect double-stranded RNA ranging from 200 to 1000 base pairs in length; the biopolymer matrix and double-­ stranded RNA are mixed to self-assemble into the nanoparticle. This nanoparticle achieves a greater effectiveness for delivering RNA inhibitory agents into insects (IPwatchdog 2015). Another example if the US8575424B2 granted in 2013 to current assignee DOWDUPONT, titled, “Production of functionalized linear DNA cassette and quantum dot/nanoparticle mediated delivery in plants”, claims methods for introducing a functionalized linear nucleic acid cassette molecule of interest into a plant cell comprising a cell wall includes use of nanoparticles. In some embodiments, the cell comprising a cell wall is a cultured plant cell. Methods include genetically or otherwise modifying plant cells and for treating or preventing disease in any plant,

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especially crop plants. Transgenic plants include a nucleic acid molecule of interest produced by regeneration of whole plants from plant cells transformed with functionalized linear nucleic acid cassette molecules. This field is cross disciplinary bringing together the best minds from all fields of science and technology. Creative collaborations between physicists, biologists, chemists and agricultural scientists are facilitating the development and introduction of new bioformulations for focused delivery and application practices, with near-complete understanding of the impact of plant defence induction kinetics on application timing and placement of formulated products at the desired sites and lead to enhanced field performance, reducing inconsistency of field performance thereby encouraging the farmers to implement the evolving technologies for the betterment of our humankind. Scientists and academicians from the emerging economies and the developed economies over the last two decades have been intensely engaged in nano-­ biopesticide-­related R&D and voluminous publishing. Surprisingly, the effective commercial output or even patenting from research groups and industries in the emerging economies has been negligible. This is an area of serious concern, and therefore there is an urgent need to integrate IPR as an integral part of the innovation value chain. Most researchers in the field of biopesticides and nano-biopesticides do not refer to patent literature either as a source of structured technical information resource or as a source of live prior art, and hence most academic work in this field lead to “re-inventing the wheel” with little or no commercial value. Clearly, the global nano-biopesticide market in the future will be in the hands of a few of early-mover the companies listed in Tables 1.1 and 1.2 in this chapter. Strategic consolidation of intellectual property assets through acquisitions, mergers, collaborations and licencing agreements is expected to dominate the business dynamics in the years to come. Limitations in patenting inventions in nano-biopesticides. At this stage, it is pertinent to revisit the TRIPS Agreement, especially the Article 27 which states: 1. Subject to the provisions of paragraphs 2 and 3, patents shall be available for any inventions, whether products or processes, in all fields of technology, provided that they are new, involve an inventive step and are capable of industrial application. Subject to paragraph 4 of Article 65, paragraph 8 of Article 70 and paragraph 3 of this Article, patents shall be available and patent rights enjoyable without discrimination as to the place of invention, the field of technology and whether products are imported or locally produced. 2. Members may exclude from patentability inventions, the prevention within their territory of the commercial exploitation of which is necessary to protect public order or morality, including to protect human, animal or plant life or health or to avoid serious prejudice to the environment, provided that such exclusion is not made merely because the exploitation is prohibited by their law.

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3. Members may also exclude from patentability: (a) Diagnostic, therapeutic and surgical methods for the treatment of humans or animals. (b) Plants and animals other than micro-organisms, and essentially biological processes for the production of plants or animals other than non-biological and microbiological processes. However, Members shall provide for the protection of plant varieties either by patents or by effective sui generis system or by any combination thereof. The provisions of this subparagraph shall be reviewed 4 years after the date of entry into force of the WTO Agreement. The patent laws in various countries do incorporate exclusion provisions as allowed by Article 27. In compliance with Article 27.2 and 3, in most national or regional patent laws, patentable subject matter is defined negatively, i.e., by providing a list of what cannot be patented. Whilst there are considerable differences between countries, the following are examples of some of the areas that are excluded from patentability in many jurisdictions: • • • • •

Abstractions and scientific theories Aesthetic creations Schemes, rules and methods for performing mental acts Substances as they naturally occur in the world Inventions the exploitation of which may affect public order, good morals or public health • Diagnostic, therapeutic and surgical methods of treatment for humans or animals • Plants and animals other than microorganisms, and essentially biological processes for the production of plants or animals other than non-biological and microbiological processes • Computer programs With specific reference to nano-biopesticides, the following aspects may act as barriers to patentability: • The possible adverse and/or unforeseen effects on the soil, plants and the environment could invoke provisions in patent laws with regard to exclusions from patentability inventions, the prevention within their territory of the commercial exploitation of which is necessary to protect public order or morality, including to protect human, animal or plant life or health or to avoid serious prejudice to the environment, provided that such exclusion is not made merely because the exploitation is prohibited by their law. • Plants and animals other than microorganisms, and essentially biological processes for the production of plants or animals other than non-biological and microbiological processes. • Substances as they naturally occur in the world. • Abstractions and scientific theories.

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Severe doubts have been raised about the phytotoxicity profiles of nano-­ biopesticides, their possible effects on the bacterial diversity, change in soil pH, effect on root length growth, number of roots, their retention in the soil thereby causing possible long-term damage, the microbes developing resistance to nanoparticles, their effect on other crops, etc are areas of grave concern. Ingestion of the nano-biopesticides by grazing animals, their leaking into the ground water system, their impact on cell biology and possibly influencing the genetic code are areas that need to be looked into with care and caution. These uncertainties may spark negative posturing towards considering inventions in nano-biopesticides as being non-­ patentable subject matter unless proven otherwise. Using naturally occurring matter (e.g. containing material from algae, lichens, bryophyta, multicellular fungi or plants or extracts thereof) in nano-biopesticides, minerals, clays, etc may be objected from being patentable unless the experiments demonstrate significant intervention from the inventors in their modifications, structuring and incorporations to differentiate them from their naturally occurring state. Further, a lot is known in traditional agricultural practices where bio-based materials have been used as pesticides for generations by communities and therefore may come on the way of patenting as prior art. In several cases, the mechanisms and modes of action of nanomaterials are not known, and there is a lot of associated hypothecation that may be considered to be abstraction and theorisation. Constant interactions between the inventors and patent experts will be essential to introduce on-course corrections in the designing of experiments to avoid the general pitfalls of inadequate enablement of the invention by “a person of ordinary skill in the art” and/or inadequate examples by way of experiments to illustrate the range of claims. Extreme care will have to be exercised whilst drafting the patent specifications and structuring the claims to avoid misinterpretation of them being understood to be within the ambit of the exclusions as “non-patentable subject matter”.

1.5  Mitigating Potential Risks New nanotechnologies including the use of nanoscale materials in the agricultural and food value chain raise pertinent questions about their potential health, safety and environmental impact. This is a subject matter for serious consideration by regulatory agencies including scientific platforms, public and private special interest organizations, industry and industry associations whilst adopting appropriate regulations to ensure responsible development and exploitation of these wonder-­ material and technologies to their full potential. Major collaborative activities are with active participation of all stakeholders and government, with possible open sharing of data related to health and safety. The challenge is to finally set the standards for testing & monitoring methods, health, safety and environment. Another area that will need due consideration will be the view that competition authorities would take whilst evaluating consolidation of resources in the marketplace by way of IPR transactions between various industry partners, technology transfers,

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collaborations mergers, acquisitions, creation of joint ventures, pricing of products and services. Business strategies will have to be designed and tailored to balance responsible use of IPR with in the innovation and business value chain.

1.6  Conclusion Nano-biopesticides is a fast growing commercially attractive proposition in view of a positive shift to green and precision agricultural practices. Being innovation-led, intellectual property rights by way of ownership of inventions, transactions of IP at all stages of the business value chain will play a major role in the market dynamics. Real-time care will have to be taken to ensure that inventions are focused with clear objectives based on patent-based technology landscapes, diligently planned and executed so that at the stage of drafting, filing, prosecuting IP applications, all possible aspects that might come on the way of obtaining the grant of the IPR are taken care of well ahead of time. Further, nano-biopesticides are now becoming fiercely competitive, and therefore one will have to be sensitive to possible infringements of others’ IPRs. In addition to inventing, one will also have to ensure that there is freedom to operate in the territories of business interests and hence making IPR an integral part of the innovation-value chain is an imperative. Acknowledgement  The author is grateful to Dr. Murari Venkataraman and Dr. George Koomullil of Relecura Technologies Pvt Ltd, Bangalore, for acceding to the author’s request to conduct the patent literature searches in nano-biopesticides, generously providing the necessary data including the permission for use in this chapter.

References Athanassiou CG, Kavallieratos NG, Benelli G, Losic D, Rani PU, Desneux NJ (2018) Nanoparticles for pest control: current status and future perspectives. Pest Sci 91:1–15 Duhan JS et al (2017) Nanotechnology: the new perspective in precision agriculture. Biotechnol Rep 15:11–23. https://doi.org/10.1016/j.btre.2017.03.002 Gogos A, Knauer K, Bucheli TD (2012) Supporting information: nanomaterials in plant protection and fertilization: current state, foreseen applications, and research priorities. J Agric Food Chem 60(39):9781–9792 IP Australia (2016). https://www.ipaustralia.gov.au/tools-resources/publications-reports/patentanalytics-report-agricultural-nanomaterials IPwatchdog (2015). http://www.ipwatchdog.com/2015/10/08/the-future-of-agricultural-pestcontrol-is-biopesticides-iot-insect-monitoring-systems/id=62282/ Kumar S, Nehra M, Dilbagh N, Marrazza G, Hassan AA, Kim KH (2019) Nano-based smart pesticide formulations: emerging opportunities for agriculture. J Control Release 294:131–153 Narayanan A, Sharma P, Moudgil BM (2013) Applications of engineered particulate systems in agriculture and food industry. KONA Powder and Particle J 30:221–235 Nuruzzaman MD, Rahman MM, Liu Y, Naidu R (2016) Nanoencapsulation, Nano-guard for pesticides: a new window for safe application. J Agric Food Chem 64:1447–1483

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Transparency Market Research (2017) Biopesticides market (Product – bioinsecticides, biofungicides, bioherbicides, bionematicides, and PIP; application – cereals and grains, oilseeds and pulses, fruits and vegetables, nurseries, and turf  – global industry analysis, size, share, volume, growth, trends, and forecast 2017–2025; https://www.transparencymarketresearch.com/ biopesticides-market.html Uno Fortune Inc. https://unofortune.en.taiwantrade.com/product/nano-5-3-in-1-natural-mucilageorganic-fertilizer-294455.html# WIPO. www.wipo.int/publications and https://www.wipo.int/edocs/pubdocs/en/wipo_pub_917_1. pdf WTO. https://www.wto.org/english/thewto_e/whatis_e/tif_e/agrm7_e.htm

Chapter 2

Application of Nanomaterials in Plant Disease Diagnosis and Management Mujeebur Rahman Khan, Tanveer Fatima Rizvi, and Faheem Ahamad

Contents 2.1  I ntroduction 2.2  A  pplication of Nanomaterials in the Detection and Diagnosis of Plant Diseases 2.3  Application of Nanoparticles and Nanomaterials in Plant Disease Management 2.3.1  Effect of Nanoparticles on Plant Pathogenic Fungi 2.3.2  Effect of Nanoparticles on Bacteria 2.4  Conclusion References

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2.1  Introduction Increasing human population has put tremendous pressure on natural resources for production of food material adequate to feed the existing and future burgeoning populations of humans and animals. Pests and diseases destroy around 30–40% of crop produce during preharvest and postharvest stages. An immediate increase of 15–20% in the crop productivity in Asia and Africa may be achieved without increasing the cultivation areas, if appropriate plant protection strategies are adopted as a precautionary as well as curative measure. In this direction, innovative plant protection approaches may play a key role in increasing food production. Since pesticide application is costly as well as hazardous, their application should be avoided or minimized to make the plant protection a low cost, farmer-friendly, and ecologically sustainable practice. Nanotechnology is one of the few highly

M. R. Khan (*) · T. F. Rizvi · F. Ahamad Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh, India © Springer Nature Switzerland AG 2019 K. A. Abd-Elsalam, R. Prasad (eds.), Nanobiotechnology Applications in Plant Protection, Nanotechnology in the Life Sciences, https://doi.org/10.1007/978-3-030-13296-5_2

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innovative and promising approaches, which may offer a reliable solution to the existing and future food production issues globally. Nanotechnology has tremendous potential to improve crop productivity (Gruère et  al. 2011), protect plants (Pérez-de-Luque and Hermosín 2013), monitor/detect plant diseases (Frewer et al. 2011), increase global food production (Biswal et al. 2012), enhance food quality (Sonkaria et al. 2012), and reduce waste production for sustainable food production (Prasad et al. 2014). The nanoparticles (NPs) have a high surface-to-volume ratio that increases their reactivity and possible biochemical activity (Dubchak et  al. 2010). For example, when 1  g gold is converted into nanoscale, the particles may cover an area of 100 km2. Gold nanoparticles (2.5 nm) melt at much lower temperatures (~300 °C) than a gold slab (1064 °C; Buffat and Borel 1976). A material after conversion into nanoform (1–100 nm) expresses some new properties with effects different from the macroform. For example, silver or gold is not toxic to microorganisms, but its nanoform inhibits microbial growth (Wang et  al. 2011; Sofi et  al. 2012). For having ultra-small size, NPs have great scope and application for use in detection and diagnosis of pathogens as well as in the management of plant diseases.

2.2  A  pplication of Nanomaterials in the Detection and Diagnosis of Plant Diseases The accurate detection and diagnosis of plant pathogens is an important step in proper disease management, and it helps in executing timely application of pesticides for proper management of the disease field. Nanoparticles can be used as biomarkers or as a rapid diagnostic tool for detection of plant pathogenic bacteria (Boonham et al. 2008), viruses (Yao et al. 2009), and fungi (Chartuprayoon et al. 2010). Nanoparticles can either be directly modified for use in pathogen detection or for a diagnostic tool to detect specific chemicals synthesized under a diseased condition. Lopez et al. (2009) reported that nanochips are microarrays that contain fluorescent oligo capture probes through which the hybridization can be detected. The nanochips are known for their high sensitivity and specificity in detecting single nucleotide change in bacteria and viruses. Plants generally respond to pathogenic infections through physiological changes such as induction of systemic defense, which is thought to be regulated by jasmonic acid, methyl jasmonate, and salicylic acid (Khan and Haque 2013). A sensitive electrochemical sensor, using modified gold electrode with copper nanoparticles to monitor the levels of ascorbic acid, salicylic acid, etc. in the plant or seeds, can be used to detect the pathogen. Wang et  al. (2011) successfully and accurately measured salicylic acid using the gold electrodes with CuNP sensor. Researches on similar sensors and sensing techniques are in progress, and sensors for detecting pathogens and their by-products to monitor physiological/biochemical changes in the host plant are likely to be available in market soon at affordable and economic costs.

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Nanoparticles display fascinating electronic and optical properties and can be synthesized using different types of materials for sensing applications (Shipway et al. 2000). For biosensing application, the limit of detection and the overall performance of a biosensor can be greatly improved by using nanomaterials. Various types of nanostructures have been evaluated to develop a biosensor for recognition of live cells, tissues, microbes, etc. The immobilization of the biorecognition element, such as DNA, antibodies, enzymes, etc., can be achieved using various approaches, including biomolecule adsorption, covalent attachment, encapsulation, or a sophisticated combination of these methods (Fang and Ramasamy 2015). The nanomaterials used for biosensor construction include metal and metal oxide nanoparticles, quantum dots, carbon nanomaterials, such as carbon nanotubes and graphene as well as polymeric nanomaterials (Fang and Ramasamy 2015). Nanoparticles can be integrated with other biological materials such as antibodies to construct immunosensors to detect bacterial infections. Yao et al. (2009) successfully prepared silica nanoparticle biomarker using silica NPs along with antibodies and developed silica NP probe to detect bacterial spot disease-causing bacteria, Xanthomonas axonopodis pv. vesicatoria. Shipway et  al. (2000) constructed gold nanoparticle-based optical immunosensors for detecting Karnal bunt disease in wheat. Lopez et al. (2009) reported that the nanochips made of fluorescent oligo probes can detect single nucleotide change in the bacteria and viruses with high sensitivity and specificity. Similarly, nano-gold-based immunosensor has been prepared to detect Karnal bunt causing pathogen, Tilletia indica in wheat (Singh et al. 2010). The use of the above sensors in the seed certification and plant quarantine may prove highly effective and accurate in detecting the microbial infections. Quantum dots (QD) due to their unique and advantageous optical properties have been used to prepare biosensors for disease detection and diagnosis using fluorescence resonance energy transfer (FRET) mechanism (Frasco and Chaniotakis 2009; Algar and Krull 2008). The QD sensors describe energy transfer between two light-­ reactive molecules. Rad et al. (2012) reported that QD-based sensors were found successfully effective in detecting the witches’ broom disease of lime caused by Candidatus Phytoplasma aurantifolia. The QD sensor showed a high sensitivity and specificity of 100% and a high degree of detection of Ca. P. aurantifolia. Safarpour et al. (2012) further observed that QD-FRET was effective in detecting the disease vectors. For example, Polymyxa betae is a vector of beet necrotic yellow vein virus (BNYVV), which is the most destructive disease in sugar beet. The vector was successfully detected using the QD-FRET-based sensor. Besides QD-based sensors, other novel nanomaterials for sensor fabrication have been researched to attain high sensitivity and detection limits (Kuila et  al. 2011; Pérez-López and Merkoçi 2011; Shiddiky and Torriero 2011). Gold nanoparticles possess a very high degree of electroactivity and electronic conductivity for electron transfer (Cao et  al. 2011; Mandler and Kraus-Ophir 2011). Umasankar and Ramasamy (2013), exploiting the characteristics of AUNPs, developed Au NP-based electrochemical sensor for detecting a plant disease. According to them, the application of AuNP-modified electrode successfully achieved electrochemical

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detection of methyl salicylate, a key plant volatile organic compound (VOC) released by plants during infections. Nanoparticles of semiconductive metal oxides also possess potential for detecting the VOCs and may be used commercially due to their low cost, suitability for electron conduction for amperometric signal, and the ease at which to obtain a desired size and shape. Fang et al. (2014) have demonstrated that application of metal oxide nanoparticles such as SnO2 and TiO2 may effectively detect VOC, such as p-ethylguaiacol produced by infected strawberry, with a detection limit for nanomolar concentration. Besides detecting plant VOC, nanoparticles can also detect compounds released by the pathogens. Boonham et al. (2008) and Chartuprayoon et al. (2010) have reported that using the nanoparticle-based ­amperometric biosensors, a range of plant pathogenic bacteria, viruses, and fungi can be detected. . Zhao et al. (2008) have reported that during the recent years, sensor-based approaches with transduction mechanisms such as optical, electrochemical, and mass sensitive measurements have offered an accurate and reliable technology for detection of microorganisms. Gold nanoparticle (gold NP)-based colorimetric biosensing assays have recently attracted considerable attention in detection and diagnosis of microorganisms, because of their simplicity and versatility. The DNA-AuNP probes as a new generation of biosensorbased detection tools offer a promising technology for precision in biological sciences. Wang and Ma (2009) have reported that for having unique optical properties, gold NPs have been extensively explored as probes for sensing/imaging a wide range of analytes and targets such as heavy metallic cations, nucleic acids, proteins, cells, etc. The AuNPbased probes have been successfully used to detect bacterial infections (Thompson 2004; Basu et al. 2004; Gill et al. 2008; Nelson et al. 2001). Castañeda et al. (2007) have shown that thiols and other functional groups, which have the capacity to strongly interact with gold NPs, could be exploited to immobilize DNA strands on gold NPs. According to Sato et al. (2003), two major factors that play important roles in high sensitivity of AuNP-based probes, cross-linking of AuNPs and elevating salt concentration are critical for aggregation of the NPs. The aggregation of AuNPs leads to the shift in absorbance peak toward a longer wavelength. As a result, the solution turns purple. Storhoff et al. (1998) observed that formation of a larger aggregate of AuNPs is required to attain a detectable colorimetric shift, which is essential to augment the sensitivity of AuNP-based probes. They employed AuNP probes for colorimetric detection of DNA molecules extracted from P. syringae pathovars-specific primers designed to amplify a sequence located at a region of bacterial genomes called pathogenicity islands. AuNP probes were added to the PCR-amplified fragment containing the hrcV gene. This gave rise to the formation of a polymeric network of DNA-AuNPs with a concomitant red-topurple color change, which was used as P. syringae strain indicator. Vaseghi et al. (2013) have reported that DNA-gold nanoparticle probes can successfully detect the infection by Pseudomonas syringae pathogenic varieties (pathovars). The P. syringae pathovars are significant gram-negative pathogens among phytopathogenic bacteria, causing a variety of blights, streaks, and spot diseases in many important agricultural crops, including tomato, soybean, rice, and tobacco (Horst 1990). To prevent the incidence of these pathovars, appropriate methods should be employed. Conventional detection of P. syringae has been based on bacterial

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isolation followed by pathogenicity tests, biochemical or serological techniques, etc. which take a lot of time. However, DNA-AuNP biosensors may detect and confirm the infection precisely within a few minutes (Vaseghi et al. 2013).

2.3  A  pplication of Nanoparticles and Nanomaterials in Plant Disease Management Limited information on pathogen-suppressive effects of nanoparticles is available with regard to field application. However, considerable work has been done in in vitro condition. Researches have reported the antimicrobial property of different nanoparticles, for example, AgNPs against various harmful microorganisms (Chambers et  al. 1962). Suppressive effect of silver ions on E. coli and Staphylococcus sp. was recorded by Berger et al. (1976).Both fungi and bacteria have also been reported to be inhibited due to NP treatments (Khan and Rizvi 2014, 2017; Khan et al. 2019). The effects of NPs on fungi and bacteria are being presented here separately.

2.3.1  Effect of Nanoparticles on Plant Pathogenic Fungi 2.3.1.1  Silver Nanoparticles Silver NPs may inhibit plant pathogens in various ways (Clement and Jarret 1994), and the disease management achieved so may be effective and safer than chemical fungicides (Park et al. 2006). Antifungal activity of different forms of silver nanoparticles has been recorded on ambrosia fungus, Raffaelea sp. (Kim et al. 2009), and sclerotium-forming fungi (Min et al. 2009). The effects were, however, dose- and treatment-dependent. Various forms of silver ions and nanoparticles are also known to suppress plant pathogenic fungi, such as Bipolaris sorokiniana and Magnaporthe grisea (Jo et al. 2009). The NP treatments significantly suppressed the colonization of the above fungi. Effective concentrations of the AgNPs in inhibiting the colonization by 50% (EC50) were higher for B. sorokiniana than for M. grisea. Growth chamber inoculation assays further confirmed that both ionic and nanoforms of silver significantly reduced the diseases caused by B. sorokiniana and M. grisea on perennial ryegrass, Lolium perenne. The NP application 3 h before spore inoculation was found highly effective, but the efficacy decreased when NPs were applied 24 h after inoculation. Kasprowicz et al. (2010) recorded a significant reduction in the mycelial growth of Fusarium culmorum, when spores were incubated with silver nanoparticles. The severity of diseases caused by Golovinomyces cichoracearum or Sphaerotheca fusca on cucumber, melons, pumpkins, etc. under field condition was suppressed when different concentrations of AgNPs were applied before

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and after outbreak of the diseases (Lamsal et  al. 2010). The 100  ppm AgNPs when applied before and after the outbreak of the disease provided greatest control of the diseases. The AgNP application resulted in maximum inhibition in the growth of fungal hyphae and conidial germination in vivo. The antifungal effects of three different AgNPs were examined against a range of plant pathogenic fungi, namely, A. ­alternata, A. brassicicola, Botrytis cinerea, Cladosporium cucumerinum, Corynespora cassiicola, Cylindrocarpon destructans, Didymella bryoniae, Fusarium oxysporum f. sp. cucumerinum, F. oxysporum f. sp. lycopersici, F. oxysporum, F. solani, Glomerella cingulata, Monosporascus cannonballus, Pythium aphanidermatum, Pythium spinosum, and Stemphylium lycopersici in vitro (Kim et al. 2012). The AgNPs/PVP have also been reported suppressive against different yeasts and molds, such as Candida albicans, C. krusei, C. tropicalis, C. glabrata, and Aspergillus brasiliensis. The hybrid materials showed strong antifungal effects against the above microbes (Bryaskova et  al. 2011). The treatment with silver nanoparticles greatly suppressed plant pathogenic fungi, viz., Alternaria alternata, Sclerotinia sclerotiorum, Macrophomina phaseolina, Rhizoctonia solani, B. cinerea, and Curvularia lunata (Krishnaraj et al. 2012). They found that 15 mg/l concentration of NPs greatly inhibited the activity of all the tested fungi. 2.3.1.2  Zinc Nanoparticles Zinc have been widely used in conventional pesticides to control pests and diseases. The nanoform, zinc oxide nanoparticles (ZnO NPs), has also been studied in various antifungal studies, which show that ZnO NPs possess substantial potential to suppress the growth of fungal pathogens (Khan et  al. 2019). Antifungal activity of ZnO NPs on pink disease of coffee caused by Erythricium salmonicolor was examined by Arciniegas-Grijalba et al. (2017). Two types of synthesized samples of ZnO NPs at different concentrations were used to determine inactivation of the mycelial growth of the fungus. The inhibitory effect on the growth and morphological change, i.e., thinning of the fibers of the hyphae and a clumping tendency of the fungus, was observed at 9 mmol L−1 concentrations of zinc acetate with high-resolution optical microscopy (HROM) and TEM. Different changes in the fungal ultrastructure, i.e., liquefaction of the cytoplasmic content, making it less electron-dense with the presence of a number of vacuoles and significant detachment of the cell wall, were observed. He et al. (2011) reported antifungal activity of ZnO NPs against Botrytis cinerea and Penicillium expansum at 12 mmol l−1. The ZnO NPs at a concentration of 3 mmol l−1 significantly inhibited the mycelial growth and colonization of the above two plant pathogenic fungi. The fungus, P. expansum, was found more sensitive to the treatment with ZnO NPs than B. cinerea.

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2.3.1.3  Sulfur Nanoparticles The elemental sulfur is a chemical that has been in wide use in pesticide formulations since ancient times. The sulfur in nanoform (SNPs) also possesses substantial fungicidal activity. The efficacy of SNPs against two fungal pathogens, Fusarium solani and Venturia inaequalis causing early blight and apple scab diseases, respectively, was examined by Rao and Paria (2013). The sulfur NPs (30 nm) were found more effective than the bigger particles in inhibiting the fungal growth. Microscopic study revealed that the fungicidal effect was mainly because of the deposition of particles on the cell wall and subsequent damage of the fungal hyphae and spores. Park et al. (2006) examined the effect of silica-silver nanoparticles against Botrytis cinerea, Rhizoctonia solani, and Collectotrichum gloeosporioides and reported suppression of the fungal growth. 2.3.1.4  Copper Nanoparticles Copper nanoparticles have been an important ingredient of chemicals that have been in use since 1885, when Millardt discovered Bordeaux mixture to control downy mildew of grapes in France (Nene and Thapliyal 1979). A number of fungicide formulations in current use also contain copper salts (Nene and Thapliyal 1979). Extremely reactive hydroxyl radicals are produced by Cu fungicides, which cause damage to lipids, DNA, proteins, and other bio-molecules of an array of microorganisms, resulting in the distinct suppression of the disease (Borkow and Gabbay 2005). Nanocopper particles suspended in water have been used since at least 1931 in a product known as Bouisol to control the diseases in grapes and other fruits (Hatschek 1931). Cioffi et al. (2004) reported antifungal activity of polymer-­ based copper nanocomposites against pathogenic fungi. CuNPs inhibited the colonization of plant pathogenic fungi, Alternari alternata, Fusarium oxysporum, Curvularia lunata, and Phoma destructiva (Kanhed et  al. 2014). Bramhanwade et al. (2016) reported that treatment with CuNPs significantly suppressed the colonization of wilt-causing fungi, F. culmorum, F. equiseti, and F. oxysporum. Ponmurugan et al. (2016) investigated the significance of green way of copper nanoparticles synthesis, which provided a simple, fast, and efficient method. In his work, the copper nanoparticles were prepared via a biological reduction method at a high concentration with good stability. Encouraging results were obtained when sprayed on red root-rot infected tea plants. Giannousi et al. (2013) observed that when Cu-based nanoparticles (11–25  nm) at concentration much lower than the commercial formulations were applied on tomato, the infection by Phytophthora infestans was controlled effectively. Similarly, Brunel et al. (2013) reported that Cu with chitosan complex nano-gels effectively controlled F. graminearum.

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2.3.2  Effect of Nanoparticles on Bacteria There are limited investigations which have examined the effect of nanoparticles on plant pathogenic bacteria (Khan et al. 2016). However, considerable information is available on the effects of nanomaterials on human pathogenic bacteria. The effects of nanoparticles on bacteria are summarized below. 2.3.2.1  Silver Nanoparticles The potential use of AgNPs and their utilization in various applications, particularly antibacterial substances in food packaging and food preservation, besides phytopathogenic bacteria, are well documented (Patra and Baek 2017). Bryaskova et al. (2011) examined the antibacterial activity of AgNPs/PVP (hybrid materials based on polyvinylpyrrolidone with silver nanoparticles) against three different groups of bacteria, Staphylococcus aureus (gram-positive bacteria), E. coli (gram-negative bacteria), P. aeruginosa (nonferment gram-negative bacteria), as well as against spores of Bacillus subtilis. The AgNPs were found highly inhibitory to gram-­ positive bacteria, such as E. coli, P. aeruginosa, and S. aureus (Guzman et al. 2009). A study conducted by Mishra and Sharma (2017) examined the effect of AgNPs against S. aureus, E. coli, and Proteus vulgaris and showed the highest inhibition of P. vulgaris colonization and lowest of S. aureus. Moderate inhibitory effect of AgNPs on the colonization of B. cereus, Listeria monocytogenes, Staphylococcus aureus, E. coli, and Salmonella typhimurium were recorded (Patra and Baek 2017). When AgNPs were mixed with antimicrobial chemicals, the inhibitory effect of the combined material was greater. 2.3.2.2  Zinc Nanoparticles The antibacterial efficacy of ZnO NPs has been examined on a number of microbes (Khan et al. 2019). Jayaseelan et al. (2012) reported that zinc nanoparticles at 25 ng/mL severity inhibited the colonization of P. aeruginosa in vitro. In another study, ZnO NPs substantially suppressed the colonization of B. subtilis, E. coli, S. aureus, and P. aeruginosa (Yousef and Danial 2012). Xie et al. (2011) reported extreme sensitivity of Campylobacter jejuni to the treatments with ZnO nanoparticles. Scanning electron microscopy examination revealed that the majority of the cells transformed from spiral shapes into coccoid forms due to exposure to 0.5 mg/ml of ZnO nanoparticles for 16 h. These morphological changes in the cells of C. jejuni were due to extreme sensitivity of the bacterium to stress conditions. These coccoid cells were found by ethidiummonoazide-quantitative PCR (EMA-qPCR) to have a certain level of membrane leakage (Xie et al. 2011).

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The concentration and particle size have been largely found to be directly correlated with the antibacterial activity ZnO NPs. Researches have shown that larger surface area and higher concentrations of zinc NPs accordingly suppressed the bacterial colonization (Zhang et al. 2007; Peng et al. 2011). Jeng and Swanson (2006) reported that increasing concentrations of ZnO NPs caused greater bacterial cell death. This apparently disrupted mitochondrial function, stimulating lactate dehydrogenase leakage, and changing the morphology of the cell at concentrations of 50–100 mg L−1. Yamamoto (2001) examined the influence of ZnO NPs (100–800 nm) on S. aureus and E. coli. It was found that antibacterial activity of ZnO NPs on S. aureus and E. coli increased with the decrease in the particle size (Zhang et al. 2007; Padmavathy and Vijayaraghavan 2008). ZnO NPs of smaller sizes can easily penetrate into bacterial membranes due to their large interfacial area, thus enhancing their antibacterial efficiency (Yamamoto 2001). The dissolution of ZnO NPs into Zn2+ was reported as size-dependent, and a few studies have suggested that this dissolution of Zn2+ to be responsible for ZnO NPs’ toxicity. 2.3.2.3  Copper Nanoparticles Although copper compounds are known for antifungal activity, this metal also possesses antibacterial property (Khan et al. 2019). Copper nanoparticles in soda lime glass powder proved substantially suppressive against gram-positive and gram-­ negative bacteria and fungi (Esteban-Tejeda et al. 2009). Effects of CuO NPs on S. aureus, B. subtilis, P. aeruginosa, and E. coli were examined in vitro, and it was found to be suppressive to all four bacteria (Azam et al. 2012). Mondal and Mani (2012) reported that nano copper effectively controlled a plant pathogenic bacteria Xanthomonas axonopodis pv. punicae that causes blight in pomegranate. The nanocopper inhibited the growth of bacterium at 0.2 ppm only, i.e., >10,000 times lower than it is usually recommended for Cu-oxychloride. Agarwala et al. (2014) examined the effects of CuO and Fe2O3 on multidrug resistant biofilm forming bacteria. They revealed that CuO NPs caused greater suppression of methicillin-resistant S. aureus (MRSA), followed by E. coli. 2.3.2.4  Other Nanoparticles Nanoforms of iron, magnesium, nickel, etc. may also suppress microbial growth. Iron oxide nanoparticles suppressed gram-positive and gram-negative pathogenic bacteria. The iron oxide nanoparticles, hematite (α-Fe2O3) of different sizes ranging from 2 to 540 nm in diameter, greatly suppressed the growth, biofilm formation, and AMP function of Pseudomonas aeruginosa (Borcherding et al. 2014). Al-Hazmi et al. (2012) reported that MgO nanowires in solid media inhibited the growth of E. coli and Bacillus sp. The antibacterial activity increased with increasing MgO nanowire concentration, and the presence of unidimensional MgO nanowires

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caused greater suppressive effects. Antimicrobial effects of NiO NPs have been examined against some bacteria, and it was found that bacterial cells treated with NiO NPs exhibited irreversible damage to the cell wall, leading to death of the cell (Rakshit et al. 2013). The nano-sized silica-silver showed antibacterial activity against plant pathogenic bacteria, 100 ppm of nano-sized silica-silver completely inhibited the bacterial colonization (Park et al. 2006). Use of Nanomaterials to Carry or Encapsulate the Pathogen-Suppressive Chemicals At the present state of knowledge and technology, pesticides are the most effective and reliable means of pest and disease management, but their applications present several ill effects, such as environmental contamination and residual toxicity in the food. Their adverse effects are mainly due to indiscriminate and overuse of pesticides. The pesticide application may prove safer if applied at a lower dose, but at the lower doses, effectiveness of pesticides is severely affected because generally 1–5% of the applied pesticide reaches to the target organism. The agrochemical industries are currently working to decrease the particle size of existing chemical fertilizers and pesticides to the nanoscale or to encapsulate the active ingredients in the nanocapsules (Khan et  al. 2019). Microencapsulation may be successfully used for hydrophobic pesticides to enhance their dispersion in aqueous media with a control on the release of active ingredient. Polymer NPs have a potential use in the nanopesticide production (Perlatti et al. 2013). The agrochemical companies have been working to produce the formulations that contain nanoparticles within the 100–250 nm size range, with greater solubility in water (Kumar et al. 2010). Nanomaterials also serve as additives, mostly for controlled release and active constituents (Gogos et al. 2012). Controlled-release (CR) formulations of imidacloprid (1-(6 chloro-3-pyridinyl methyl)-N-nitro imidazolidin-­ 2-­ylideneamine), synthesized from polyethylene glycol and various aliphatic diacids using encapsulation techniques, have been used for efficient pest management in different crops. Encapsulated formulation of fungicides zineb and mancozeb has been prepared (Sarlak et  al. 2014). The multiwall carbon nanotubes-graft-poly citric acid (MWCNT-g-PCA) hybrid material was used to encapsulate the above pesticides. The effect of encapsulated pesticide was studied on Alternaria alternata on potato dextrose agar. Results showed that nanofungicide, in contrast to bulk pesticide, had an extraordinarily superior suppressive effect on A. alternata.

2.4  Conclusion The nanomaterials possess great scope and potential for use in diagnosis and management of plant diseases. Enzyme-based biosensors coated with Au, Ag, Cu, and Ti-NPs may greatly enhance the sensitivity of diagnostic probes for plant infection detection. The nanomaterials may be used in plant disease management through two

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ways, i.e., direct application of the nanoparticles of a suitable antimicrobial chemical or by encapsulating an antimicrobial chemical by a nanomaterial. Nanomaterials, nanotubes, and nanocapsules can efficiently carry higher concentration of active ingredients of pesticides, etc. and may regulate the release of the active principle. Direct application of nanoparticles may suppress plant pathogenic fungi and bacteria. However, biosafety aspects with regard to NP accumulation as well as phytotoxic and mammalian toxic effects of the nanomaterials remain major concerns that have to be ruled out before commercial use of nanomaterials in plant disease management.

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Chapter 3

Bio-Engineered Nanomaterials for Plant Growth Promotion and Protection Naradala Jayarambabu and Kalagadda Venkateswara Rao

Contents 3.1  I ntroduction 3.1.1  Nano-Biotechnology 3.1.2  Nano-Nutrition 3.2  Green Nanoparticles for Plant Growth Promotion and Protection 3.2.1  Effect of Nano-carbon on Plant Growth Promotion and Protection 3.3  Metal Nanoparticles 3.3.1  Cu and CuO Nanoparticles 3.3.2  ZnO Nanoparticles 3.3.3  TiO2 Nanoparticles 3.3.4  Silver Nanoparticles 3.3.5  CeO2 Nanoparticles 3.3.6  SiO2 Nanoparticles 3.3.7  Gold Nanoparticles 3.3.8  Iron Oxide Nanoparticles 3.4  Conclusions References

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3.1  Introduction Nanotechnology is now a fast-growing technology available to meet our ever-growing needs. Nanotechnology is about things, whether it be making things that are smaller, faster, or stronger, or making something additional, or making machines that will lead to new manufacturing paradigms. Three factors that define nanotechnology are small N. Jayarambabu · K. V. Rao (*) Centre for Nano Science and Technology, IST, Jawaharlal Nehru Technological University, Hyderabad, Telangana, India e-mail: [email protected] © Springer Nature Switzerland AG 2019 K. A. Abd-Elsalam, R. Prasad (eds.), Nanobiotechnology Applications in Plant Protection, Nanotechnology in the Life Sciences, https://doi.org/10.1007/978-3-030-13296-5_3

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size, new properties, and integration of technology in devices (Abd-Elsalam and Prasad 2018); this is the key issue as in macro-scale the properties are not size dependent. In addition, implications for human and environmental health and financial and capacity challenges as they relate to developing countries are identified (Chen and Yada 2011; Gogos et al. 2012; Fakruddin et al. 2012. Nanotechnology has the potential to develop based on eco-friendly natural polymers, which in addition to being biodegradable can also be obtained from natural bio-waste (Parisi et al. 2015; Ditta et al. 2015). Nanotechnology offers, in fact, substantial prospects for the development of innovative systems and applications, and the number of products and production volume involving nanotechnology will continue to increase (Gopal et  al. 2011; Khodakovskaya et al. 2009; Ngo et al. 2014). Specifically, the application of nanoscale-based ideas ranging from 1 to 100 nm is an emerging area of nano-science and nanotechnology.

3.1.1  Nano-Biotechnology Biotechnology and nanotechnology are two of the most promising technologies of the twenty-first century. Nanotechnology (sometimes referred to as nanotech) is defined as the design, development, and application of materials/devices whose least functional makeup is on a nanometer scale. Biotechnology is concerned with metabolic and other physiological processes of biological subjects including microorganisms. These two technologies are associated; that is, nano-biotechnology can have a vital role in developing and implementing many useful tools in the study of life (Srivastava and Rao 2014; Kole et al. 2013). By leading R&D analyses, research on agricultural applications of nanotechnology has been ongoing for nearly a decade now, searching for solutions to agricultural and environmental challenges, such as sustainability, improved varieties, and increased productivity. A growing trend has been shown in both scientific publications and patents in agricultural nanotechnology for disease and crop protection (Abd-Elsalam and Prasad 2018), with the amount of sprayed chemical products being controlled by smart delivery of active ingredients, minimizing nutrient losses in fertilization and increasing yields through optimized water and nutrient management. Nanotechnology-derived devices are also being explored in plant breeding and genetic transformation: such could be a source of bio-nano-composites with enhanced physical-mechanical properties based on harvested materials, such as wheat straw and soy hulls, for bio-industrial purposes (Ditta et  al. 2015). Large companies are investigating the potential that nanotech solutions offer in the agricultural field. However, according to industry experts, agricultural nanotechnologies so far do not demonstrate a sufficiently high economic interest. Nanotech products require initial investments, which is not currently the case. Among the reasons for the difficulties of agricultural nanotechnology developments at the field level are cited regulatory issues and public opinion. Plant-based agricultural production is the basis of broad agriculture systems providing food, feed, fiber, fire (thermal energy), and fuels through advances in

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materials sciences and biomass conversion technologies. Although the demand for crop yield will rapidly increase, agricultural and natural resources such as land, water, and soil fertility are finite. Given that nanotechnology may allow for the precise control of manufacturing at the nanometer scale, novel possibilities in enhancing precision farming practices are possible. Applications of nanotechnology in agriculture are still in their infancy of use. Despite the scientific and technical knowledge achieved so far in many conditions, crop productivity potential has not been fully realized, which is attributed to low efficiency in the use of nutrients and water for crops and the stiff competition from weeds and crop pests. Nanotechnology offers a new scientific approach to break this yield barrier and may improve our understanding of the biology of different organisms. Nanotechnology can address the two needs of better yield and nutritional value by improving systems for monitoring environmental conditions and delivering nutrients or pesticides as appropriate. Also, it can offer solutions to meet the challenges of food security and of environmental remediation (Kole et al. 2013).

3.1.2  Nano-Nutrition Nano-nutrition is the application of nanotechnology for the provision of nano-sized nutrients for crop production (Mura et al. 2017). Two types of nanoparticles (NPs) have been used: biotic and abiotic. The abiotic form of nutrients or NPs is prepared from inorganic sources such as salts, but this is not safe because many of these are not biodegradable. Using nano-nutrition we can increase the micronutrients as well as macronutrients available to the plants. Nanotechnology has been applied in plant protection against insects and pests. Nanoparticles could be effective in the preparation of new formulations such as pesticides, insecticides, and insect repellents. As mentioned in later sections, nanomaterials (NMs) such as nano-silica have been used for placing targeted genes into cells, and this technique could also be used in the formulation of pesticides, insecticides, and insect repellents. Use of micronized chemical pesticides in controlling plant diseases is an age-old practice. In comparison, nano-formulation of these chemicals is a recent introduction to the field of plant protection.

3.2  G  reen Nanoparticles for Plant Growth Promotion and Protection Synthesized metals and metal oxide nanoparticles or nanomaterials have wide implications in agricultural crop production, and many studies have reported their positive effect on various crops. Understanding the interactions between engineered nanomaterials and plants is crucial. The impact of bio-nanotechnology on the environment and agriculture has a focus on toxicity concerns, plant disease treatment, and genetic engineering (Figs. 3.1 and 3.2).

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Fig. 3.1  Bio-nanoparticle treatment of seeds of mung bean and maize before seed germination

Fig. 3.2  Bio-nanoparticle treatment of seeds of tomato and mirchi before seed germination

3.2.1  E  ffect of Nano-carbon on Plant Growth Promotion and Protection Use of multiwalled carbon nanotubes (CNTs) with the cells of tomato seedlings resulted in significant changes in total genes. The tomato plants showed some altered expression of groups of genes in the roots and leaves in a medium supplemented with CNTs. CNTs can lead to the activation of many stress-related genes including the gene for water channel protein when exposed to tomato seeds. The expression of the water channel gene in CNT-exposed roots and leaves can impact the observed phenomena of activation, growth, and enhanced germination of tomato seedlings in a medium with CNTs (Srivastava and Rao 2014).

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Water-soluble multiwalled carbon nanotubes showed effects in a concentration-­ dependent manner on wheat, maize, peanut, and garlic seedlings. It is also said that CNTs can penetrate thick seed coats and support water uptake inside the seeds. Studies suggest that lower concentrations (50  mg/ml−1) are beneficial as these increase growth indices and the water content of the root is in direct contact with the medium. CNTs have positive effects on seed germination by increasing their growth with higher biomass production for the plants. The CNTs can penetrate through the seed coat, promoting the seed germination process. Exposure of tomato seeds to CNTs resulted in improved germination and seedling growth rate. Fullerenol accumulation in plant tissues and cells of root, stem, petiole, leaf, flower, and fruit at particular concentrations is a factor of increase in biomass yield, fruit, and phytomedicine content in fruits. The concepts and strategies of nano-biotechnology could also be utilized for validation and exploitation in other crops for augmentation of yield and amelioration of quality related to food, feed, fiber, fuel, aesthetics, and health (Parisi et al. 2015). The continuous use of CNT is nontoxic, and the growth rate of the plants studied here increased several times in the presence of CNTs. A key implication of CNTs could be for optimum water transport with larger water uptake to prevent water loss in the fields. The CNTs in plants can be important in arid agriculture areas where the supply of water is crucial and requires maximum conservation and usage (Husen and Siddiqi 2014). 3.2.1.1  Effect of Fullerene It was found that treatment of bitter melon seeds with fullerene increased the yield by 112–128%. The greatest biomass increase was as much as about 54%, confirming the absorption and translocation of fullerol in plants. All the cucurbitacin-B (74%), lycopene (82%), charantin (20%), and insulin (91%) were found to increase control. 3.2.1.2  E  ffects of Single-Walled Carbon Nanotubes (SWCNTs) and Multiwalled Carbon Nanotubes (MWCNTS) Onion and cucumber plants were investigated with functional SWCNTs. Root elongation was observed with the formation of sheets of carbon nanotubes. Contradicting reports have been published regarding the influence of carbon nanotubes on various plants. Khodakovskaya et al. (2013) have shown, from their experiments on similar plants, that increased water uptake by seeds increased their germination. Treatment of maize seedlings with MWCNTs affected the germination process in a concentration-­ dependent manner, such that lower concentrations are beneficial (Tripathi et al. 2011). At a concentration of 20 mg/l, pristine MWCNTs increased growth indices and the water content of morphological structures, mostly the root, which was in contact with the medium. The effect was more marked than for the shoots (at 20 mg/l) (Tiwari et al. 2013). However, for the same water content the

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length was greater for the MWCNT plants; the low dose of the MWCNT benefited the physiology in more ways than only water delivery. It can be concluded that pristine MWCNTs at low concentrations benefit the growth of maize seedlings by enhancing water content, nutrient transport, and biomass. Growth of ryegrass and tomato has been shown to be augmented by carbon nanotubes. These nanotubes are also helpful in the slow release of pesticides. Carbon/fullerene nanotechnology is a rapidly growing area of research finding use in plants, medicine, and engineering. Carbon nanotubes (both single-wall and multiwall) can penetrate the seed coat and plant cell wall, depending on their size, concentration, and solubility. The penetration of CNTs into the plant system can change metabolic functions, leading to an increase in biomass and yield of fruits and grain. The addition of CNTs to an agar medium accelerated the process of seed germination and significantly shortened germination time. Germination percentage rates during the following days were dramatically higher for seeds treated with nanoparticles. The germination percentage for seeds that were placed on regular medium averaged 32% in 12 days and 71% in 20 days, whereas germination percentage of seeds on medium supplemented with CNTs averaged 74–82% in 12  days and 90% in 20  days. Tomato seedlings that germinated and developed on medium with different concentrations of CNTs (10, 20, or 40 g/l) exhibited a dramatic increase in vegetative biomass. Fresh weight of total biomass (leaves, stems, and roots) increased 2.5 fold for the seedlings germinated and grown on medium containing CNTs as compared with seedlings developed on standard medium. The results suggested that CNTs could significantly enhance water uptake inside tomato seeds, and that this activated water uptake could be responsible for the faster germination rates and higher biomass production for plants exposed to the CNTs.

3.3  Metal Nanoparticles Nanoparticles of Fe, Co, and Cu synthesized by the aqueous solution reduction method, with particle size from 20 to 60  nm, were used to treat soybean seeds, which responded variably depending on concentration. Nano-crystalline cobalt exhibited the best biological effects on soybean growth and development, more than 2.4 fold, and crop yield was about 16% greater in comparison with control. The studies showed that low concentrations of nano-crystalline metals can be used in various agriculture applications.

3.3.1  Cu and CuO Nanoparticles Concentrations of CuO nanoparticles have shown benefits on the growth of soybean and chickpea seedlings, with maximum growth at 100 ppm for soybean seedlings and 60  ppm for chickpea seedlings. CuO nanoparticles increased the effective

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growth of soybean and chickpea seedlings at certain optimal concentrations and inhibited growth beyond this concentration. In particular, the exposure of plants to nanomaterials and the impacts of such materials on plant systems could open a new direction for research in nanotechnology (Adhikari et al. 2012). The potential effect of engineered NPs on soil organisms was measured using a plant assay. When Phaseolus radiatus and Triticum aestivum were exposed to concentrations of Cu NPs, the amount of cupric ions released from the Cu NPs had negligible effects. The Cu NPs also were bioavailable, as CuO NPs can be taken up, transported, and biotransformed by rice plants under hydroponic conditions (Peng et al. 2015; Saharan et al. 2015). CuO NPs can move into the root epidermis, exodermis, and cortex, and finally reach the endodermis, but it is difficult for CuO NPs to pass through the Casparian strip. Cu-chitosan NPs can be used as antifungal agents against early blight and Fusarium wilt of tomato. Studies show that Cu-chitosan nano-formulation could be a new development for the generation of chitosan-based bio-nanopesticides.

3.3.2  ZnO Nanoparticles ZnO nanoparticle treatment of peanut seeds improved the germination, root growth, shoot growth, dry weight, and pod yield. The results emphasize that applying nanoscale nutrients to the crops either through seed dressing or by foliar application at much decreased doses will obtain the desired results (Prasad et al. 2012; Mahajan et al. 2011). ZnO NPs have shown increase in root and shoot growth of gram seedlings and mung seedlings, However, above certain concentrations the growth of roots and shoots was found to decline. For mung seedlings, the best growth response for root (42.03%, p = 0.0498) and shoot (97.87%, p = 0.0444) was observed at a concentration of 20 ppm above control. Similarly, for gram seedlings, the dose of 1 ppm promotes significant increase in growth of roots (53.13%, p = 1.125 × 10−7) and shoots (6.38%, p = 0.026) as compared to control. The biomass production in shoots of gram and mung plants was found to be in root and shoot length for corresponding nano-ZnO treatment. For nano-ZnO at 20 ppm treatment, mung seedlings showed 40.89% increase in root biomass and 76.04% increase in shoot biomass over control. In gram seedlings at 1 ppm treatment, 37.15% increase in root biomass and 26.61% increase in shoot biomass were observed. The increase in biomass at certain concentrations suggests the optimal dose limit for the growth of mung and gram seedlings (Mahajan et  al. 2011). At treatment of 20 ppm and 1 ppm concentration doses of nano-ZnO for mung and gram, respectively, the root developed very well with the usual threetissue system (epidermis, cortex, vascular cylinder) as observed in the control, which indicates no adverse effect of nano-ZnO particles on the root architecture. In H2O2 generation induced by Fe@ZnO NPs in roots, stems, and leaves in the present study, only roots at 500 mg/kg treatment showed a 50% decrease in H2O2 compared to all other results. Lower H2O2 in roots signifies less production of

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reactive oxygen species (ROS), leading toward less stress in green pea roots. In addition, H2O2 generation in stem and leaf was unaffected by Fe@ZnO treatments. However, bare ZnO NPs showed overproduction (61%) of leaf H2O2 at 500 mg/kg treatment. Lower ROS production, of lesser stress level, may be attributed to lower Zn bioaccumulation in all the plant tissues in doped-ZnO NP treatments, compared to that of bare NPs (Mukherjee et al. 2014; Jhansi et al. 2017; Jayarambabu et al. 2014).

3.3.3  TiO2 Nanoparticles Canola seeds treated with TiO2 NPs at higher concentrations showed improved germination and root growth. The results point to the beneficial use of nanomaterials in agriculture, especially in canola, one of the main sources of livelihood in certain parts of the world. It was demonstrated that nano-TiO2 treatment at the proper concentration upgrades the germination of aged spinach seeds and increases its vigor.

3.3.4  Silver Nanoparticles The use of silver nanoparticles (SNPs) for stimulation of germination, early seedling growth, and biochemical compounds of Triticum aestivum seed is possible. SNPs increased the rate and percentage of germination of Triticum aestivum seed, and also increased the roots, whereas MF treatment decreased these factors (Salama 2012). Nanoparticles improved seed germination parameters, that is, germination percentage, speed of germination, and coefficient of germination, except mean germination time. Some reports suggested that biogenic nanoparticles promoted seed germination in several plant species. Positive contribution of silver nanoparticles in increasing the germination percentage in Boswellia ovalifoliolata was also reported. Silver nanoparticles could penetrate through the seed coat and stimulate the embryo. The nanoparticles of silver showed enhancement in seedling growth by stimulating water and nutrient uptake by the treated seeds. Nanoparticles produce pores on seed coat during penetration, and these pores help in the influx of the nutrients inside the seed. Silver nanoparticles carry the nutrients along with them, which leads to the rapid germination and enhanced seedling growth rate. In contrast, significant enhancement in root and shoot length and vigor index was also reported using 25 and 50 ppm concentrations of SNPs (Najafi and Rashid Jamei 2014). Common bean and corn plants were treated with Ag NPs, which affect the growth of common bean and corn at different concentrations. The maximum effect was found at 60 ppm: the uptake of nanoparticles is dependent on the exposure concentration. This study for the first time contributed to defining a modulator role of the HO/NO signal system during the alleviation of Ag NPs or AgNO3 stress-induced Brassica nigra seed germination inhibition and monitored the upregulation of

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mRNA HO-1 expression. NO production, which is critical for plant tolerance against AgNPs or AgNO3 stress, was increased where the quenching process was revealed to be of the static and dynamic type (Falco et al. 2014). In summary, the results that chlorophyll fluorescence (ChIF) is affected by Ag NPs suggests that ChlF has great potential to be used as an analytical tool for monitoring the interaction of plants and NPs for investigating the effects of NPs on plants.

3.3.5  CeO2 Nanoparticles Nano-cerium oxide treated cucumber, alfalfa, tomato, and corn seedlings, which increased as the external nanoparticles concentration increased. At 4000 mg/l alfalfa seedlings had about 6000 mg Ce/kg dry weight and tomato about 3000 mg Ce/kg dry weight. Cucumber showed similar Ce concentrations in tissues for the 1000– 4000  mg/l treatments (about 400  mg  Ce/kg dry weight), and corn showed about 300 mg Ce/kg dry weight only at 4000 mg/l treatment (Zhao et al. 2012). The biomass production (dry weight) of 10 seedlings per plant species treated with nanoceria was investigated. The biomass of alfalfa was significantly (p