Table of Contents
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PREFACE
ACKNOWLEDGEMENTS
Introduction
Sampling
Abstract
General consideration
Tools for sampling
Sampling from aquatic and wetland
Size of the sampling area
Sampling depth
Sampling pattern
CONCLUSION
Nematode Extraction
Abstract
Extraction of soil and root nematodes
Tray Method
Sugar Flotation Method
Incubation Method
Sieving Method
Dissecting Method
Seinhorst Cyst Extraction Elutriator Method
Baunacke Method
Maceration and Filtration or Blender Method
Wood and Compost Extraction
Extraction of entomopathogenic nematode (EPN)
Nematode Isolation
Practical Procedure
Investigation on Parasitic Nematode of Mosquito
Practical Procedure
Mosquitoes Preparation in the Laboratory
Propagation of Mermithid Nematodes
Evaluation of Different Extraction Methods
CONCLUSION
Nematode Observations
Abstract
Formalin-glycerine method
TAF (Triethanolamine Formalin) Method
Slide Preparation
Benefits of Temporary Slides
Benefits of Permanent Slides
CONCLUSION
Nematode Morphological Observations
Abstract
Microscope justification
Microscope Types
Calibrating the Eyepiece Graticule
Calibrating the Microscope for Drawing
Maintenance of microscopes
Differential Interference Contrast (DIC)
Nematode Measurements
Pictorial Measurement Guide
Scanning Electron Microscopy (SEM)
Preparation of Specimens for SEM Study
CONCLUSION
Molecular Diagnosis
Abstract
DNA extraction
Isolation of Nematodes
DESS Solution
How to Transfer Nematodes to Ethanol
Genomic DNA Extraction
DNA Extraction Using the Chelex Method
Isolation of DNA from Individual Nematodes and their Connected Bacterial DNA
DNAzol kit DNA Isolation Protocol for Individual or Pooled Nematodes
NaOH Digestion of a Single Nematode
Nucleic Acid Extraction using Lysis Buffer I
DNA Extraction from Glycerine-Embedded Nematode Specimens
Lysis Buffer Method
Common Molecular Markers
Sequence Characterized Amplified Region (SCAR)
Primers (Case Study: Meloidogyne)
Restriction Fragment Length Polymorphism (RFLP)
Preparation of DNA Digestion Master Mix
Post-digestion Steps
Amplified Length Fragment Polymorphism (AFLP)
Polymerase Chain Reaction
PCR Components
DNA Template
Quality of DNA Template
Evaluating the PCR Products
CONCLUSION
Basic Bioinformatics in Nematology
Abstract
General information
Checking the sequences
Blast of the sequences
Alignment and phylogenetic analysis
What sequences should be selected?
Alignment using MEGA
Computing Pairwise Distance
Constructing Trees
Maximum Likelihood Tree
Advantages
Disadvantages
Features of a phylogenetic tree
Bootstrapping
Outgroup of phylogenetic analysis
Online tools for nematode identification
CONCLUSION
Biodiversity Analysis
Abstract
Conventional biodiversity
Sampling
Evaluation of Soil Samples
Counting of Nematodes
Biodiversity indices and data analyses
Shannon index
Simpsonʼs Index
Molecular biodiversity
CONCLUSION
Nematode Rearing and Greenhouse Studies
Abstract
Culturing of nematodes
General consideration
Globodera spp.
Heterodera spp.
Meloidogyne spp.
Radophulus spp.
Pratylenchus spp.
Aphelenchoides and Bursaphelenchus spp.
Mylonchulus spp.
Panagrolaimus spp.
Caenorhabditis spp.
Greenhouse experiments
Nematode Inoculum
Greenhouse Technique for the Evaluation of Meloidogyne
Rearing Meloidogyne Males
Reproduction Assessment
CONCLUSION
Methods and Techniques in Nematology
Authored by
Ebrahim Shokoohi
Department of Biochemistry, Microbiology
and Biotechnology, University of Limpopo
Private Bag X1106, Sovenga, 0727
South Africa
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FOREWORD I
Nematodes, also known as roundworms, are essential to soil ecology and play a crucial role in maintaining soil health. These organisms have various feeding groups, including bacterivores, fungivores, herbivores, predators, and omnivores.
Despite the herbivores or plant-parasitic nematodes, many of them, such as free-living nematodes, are beneficial for agriculture and crop production as they help in breaking down organic matter, such as dead plant material and animal waste, into nutrients that plants can absorb. In conclusion, nematodes are more than just tiny worms in the soil. They are critical to the health and productivity of agricultural ecosystems and play a significant role in ensuring global food security.
“Methods and Techniques in Nematology” is an excellent book for anyone interested in the nematology discipline. This book is specifically designed to help lecturers, researchers, farmers, and students deal with nematode problems. The book contains a variety of techniques with detailed explanations and high-quality photographs to make the learning process easier and more engaging. These photographs help bring the topic to life and make learning more enjoyable. You will be able to see the nematodes up close and appreciate their unique features. You will find all the essential information you need to understand nematodes and learn how to address any related problems. The book covers various techniques, from conventional to molecular, particularly for those wanting to start with nematology, all of which are explained in simple and easy-to-understand language.
In conclusion, “Methods and Techniques in Nematology” is an excellent resource for anyone interested in nematology. It is an easy-to-use practical guide that will help you understand nematodes and learn how to address any related problems.
Prof. Ebrahim Shokoohi's book on nematology is an invaluable academic resource that provides students with a comprehensive understanding of the subject. With years of experience teaching at various educational levels, ranging from BSc to Ph.D., Prof. Shokoohi's book is a reliable source that would appeal to a wide audience seeking a deeper understanding of the subject matter; I feel he accomplished it brilliantly.
Zafar A. Handoo
USDA, ARS, Mycology and Nematology Genetic Diversity
and Biology Laboratory, BARC-West
Beltsville, MD 20705, U.S.A.
FOREWORD II
Nematodes are remarkable organisms with amazing abilities that have made them the most common animals on the planet; however, they are rarely seen because they are microscopic. Yet people who observe them often share a common bond and instant friendship. My relationship with Dr. Ebrahim Shokoohi is just that, a brotherhood of camaraderie that is based on our mutual admiration of all things nematode. We have worked together describing the wonderful world of nematodes.
Because nematodes are so small, they are difficult to work with. For this reason, they are often neglected, but with proper techniques, they become extremely important in many different types of studies. Fortunately, Dr. Ebrahim Shokoohi has made an effort to assemble various methods of handling and investigating nematodes. They range from sampling and separating them from soil, making slides and examining them with a microscope, designing greenhouse experiments and analyzing the results, to extracting their DNA for taxonomic studies.
This book contains valuable information for anyone interested in working with nematodes because it clearly illustrates many techniques that are described in a logical, step-by-step manner, which makes it easy to follow. The nematology community will be very happy to have this valuable resource for their use.
Jonathan D. Eisenback,
Professor of Nematology
Former President of the Society of Nematologists, Virginia Tech
Blacksburg, VA 24061, U.S.A.
PREFACE
The research on nematology is significant for researchers, students, and everyone interested in this science discipline. While I was teaching nematology for about ten years, I came up with the idea of documenting the methods and techniques that would be useful for everyone. In this book, I have presented the relevant methodology within a conceptual framework of different scopes within nematology that renders technical information that is needed for students and researchers. Methods ranging from sampling to advanced techniques, including molecular surveys, are discussed in this book. The methods are presented in a way that is adaptable for the students to use in formal courses, which can also be functional when used daily by academics and educational institutions. In almost part of the book, the author's experience and the available knowledge of the expert in nematology create an opportunity to easily run the experiments and surveys.
Ebrahim Shokoohi
Department of Biochemistry, Microbiology
and Biotechnology, University of Limpopo
Private Bag X1106, Sovenga, 0727
South Africa
ACKNOWLEDGEMENTS
I am grateful to “Allah” for blessing me with a healthy mind to think about the creations and learn from nature. I am thankful to all my teachers and professors for all I have learned in nematology and plant pathology. I would like to thank Prof. Annette van Aardt for revising this book. I would also like to thank Mr. Panahi for providing some of the high-quality pictures for this book. I would especially like to thank my lovely wife for the excellent atmosphere and courage to finish and publish this book. Finally, I would like to thank my parents for the endless support, enthusiasm, and love they have given me. I dedicate my book to my beloved, my wife and son, Adrian.
Ebrahim Shokoohi
Department of Biochemistry, Microbiology
and Biotechnology, University of Limpopo
Private Bag X1106, Sovenga, 0727
South Africa
Introduction
Ebrahim Shokoohi
Department of Biochemistry Microbiology and Biotechnology, University of Limpopo, Private Bag X1106, Sovenga, 0727, South Africa
*Corresponding author Dr. Ebrahim Shokoohi: Department of Biochemistry Microbiology and Biotechnology, University of Limpopo, Private Bag X1106, Sovenga, 0727, South Africa; E-mail: ???
Nematology is an important branch of biological science that focuses on the study of a diverse group of roundworms known as nematodes. In addition, nematology plays a crucial role in agriculture. It is integral to the management of crop pests and the enhancement of agricultural productivity through the study of nematode interactions with crops. Additionally, in the medical field, nematology is vital for controlling diseases caused by parasitic nematodes. Nematodes also serve as important bio-indicators of environmental health in environmental studies, offering insights into soil quality and ecological balance. These organisms can be found in virtually all environments around the world. The term nematode has its roots in Greek, deriving from the words "nema" and "oides", which mean thread and resembling, respectively. Nematodes are an amazingly diverse group of organisms that can either be beneficial or parasitic to plants and animals alike. Generally, they have a slender body that is transparent and lacks segments, exhibiting bilateral symmetry. The study of nematology has made significant strides in the field of medicine. Notably, it has played a pivotal role in developing new antibiotics for the treatment of bacterial infections. Furthermore, nematology has been instrumental in identifying that tropical diseases like elephantiasis and ascariasis are caused by a type of nematode. Ascariasis is a parasitic infection caused by Ascaris. The disease occurs when individuals ingest food or water contaminated with the eggs of Ascaris species, typically found in soil, vegetables, fruits, and other foods. Research has revealed that Ascaris infections can potentially lead to impaired cognitive function in certain school-aged children. This underscores the importance of proper hygiene and food safety measures in preventing the spread of this disease. Several nematodes cause diseases in animals, such as fish, where they reduce the quality of the meat and pose a risk to food security. Nematodes are a type of parasite that can infect various types of fish in freshwater, marine, and brackish water environments. Some nematodes can have devastating effects on wild fish populations, leading to significant fish mortality. These parasites can infect fish in their adult stage, but their larval forms can also infect fish species after passing through birds, mammals, or reptiles that consume fish, or even through predatory fish. Certain nematodes, like Anisakis,
are zoonotic, meaning they can be transmitted to humans. Consumption of raw/undercooked infected fish meat poses a risk of infection to humans. Nematodes also pose a threat to the economic value of fish due to consumer concerns about their presence in food products. Infected fillets are often rejected, leading to increased production costs. Therefore, research on nematology brings valuable insight into the fish/animal parasites aiming to secure food.
In agricultural research, nematology plays a crucial role in providing a wide range of ecosystem services that have a significant impact on the nitrogen cycle, the ability of soil to decompose waste, and the control of pests within soil systems. Although, certain female plant-parasitic nematodes can take on a spherical shape, such as cyst and root-knot females. All plant-parasitic nematodes possess a stylet in their anterior end that helps them to pass the food through the plant cells to their body. This feature enables plant-parasitic nematodes to inject the enzymes into plant cells, which digest the food and help nematodes to develop and cause damage to plant cells. Plant-parasitic nematodes pose a significant threat to various plant types, including vegetables, trees, turfgrass, and foliage plants. They can cause extensive damage and significantly reduce crop yield. Root-knot, cyst, root lesion, spiral, burrowing, bulb and stem reniform, dagger, bud and leaf, and pine wilt disease are among the most harmful nematodes. These pests are responsible for an average loss of 12.3% annually in 40 major crops worldwide, with developing countries bearing the brunt of the losses, estimated at 14.6%, compared to 8.8% in developed nations. Plant-parasitic nematodes result in global economic losses of nearly $125 billion per year, affecting all agricultural crops. The impact of harmful nematodes on agricultural production is regularly undervalued, as their symptoms are frequently mistaken for other issues such as water stress, nutritional disorders, virus infection, soil fertility problems, or complex diseases caused by interactions of fungal/bacterial with nematodes. The severity of their impact depends mostly on the population density in the soil and roots, the cultivar susceptibility, and the ecological circumstances. Root-knot and cyst nematodes are the main destructive plant-parasitic nematodes. The implications of these misinterpretations can be significant, as they can lead to the implementation of inappropriate corrective measures, resulting in further damage to crops and reduced yields. Therefore, an accurate diagnosis of nematode infestation is critical in ensuring optimal agricultural productivity and profitability. To achieve this, it is essential to utilize appropriate diagnostic techniques to differentiate nematode symptoms from those of other conditions. This approach can help farmers and agricultural experts make informed decisions regarding the management and treatment of nematode-infested crops, ultimately leading to better outcomes for all involved.
On the other hand, beneficial or free-living nematodes play a critical role in soil health due to their contribution to soil nutrition, nitrogen fixation, and microbial balance. The soils in a hectare of all agroecosystems typically contain billions of both plant-parasitic and beneficial nematodes, which can significantly affect crop yields. In conclusion, nematology discipline is an attractive and principal field of study that plays a vital role in understanding the complex relationships between nematodes, plants, humans, animals, and their environment.
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