Methods and Techniques in Nematology

The process of sampling nematodes has become a crucial aspect of agricultural research. The accurate identification of these pests is essential, and the method of sampling is dependent on whether they are plant-parasitic or free-living. To ensure that errors are minimized and samples are reliable and representative, the sampling pattern is based on the area being studied. This can range from randomized to systematic sampling techniques. Furthermore, the final results are influenced by various factors such as the timing of sampling, depth of samples, and the total number of samples taken.

Obtaining an adequate number of nematode specimens is crucial for various studies related to biodiversity, taxonomy, and management. The extraction method chosen for the nematodes depends on the specific study being conducted. In regards to sedentary endoparasites, the roots are the primary source for nematode extraction, while for other purposes, the soil is the preferred option. It is essential to evaluate the efficiency of the extraction method used. Nematode extraction must be performed promptly after sample collection since the specimens tend to deteriorate with time. In this chapter, some of the commonly used techniques for nematode extraction are given.

Preservation of nematodes is a crucial element of research endeavors, and it can be accomplished either temporarily or permanently. In order to conduct a rapid examination, researchers may place a minute droplet of water onto a glass slide for temporary observation. However, for the purpose of long-term preservation and collection, permanent slides are requisite. Prior to studying nematodes under a microscope for taxonomic purposes, it is necessary to first kill and fix them. This method is particularly useful for taxonomic studies involving light, electron (SEM), or transmission electron (TEM) microscopy.

In the field of nematology, a variety of microscopes are utilized for distinct purposes, including standard light microscopes and stereomicroscopes. Additionally, line illustrations are essential for the taxonomical examination of nematodes. As such, it is crucial to properly calibrate and maintain microscopes to ensure accurate outcomes. This section delves into the essential duties associated with microscope usage in nematological research. Differential interference contrast microscopy (DIC) is a useful technique to create a high-quality photograph of the nematodes. In regard to taxonomy and diagnosis of nematodes, accurate measurements are of utmost importance. Each nematode family, whether they are free-living or plant-parasitic, necessitates specific measurements of particular attributes. The identification of nematodes relies on the de Man indices. This section offers visual aids for different families to showcase the significant features that must be measured. The art of capturing images of nematodes holds immense significance in the fields of taxonomy and histological research. Nematology employs numerous methods to obtain top-notch visuals for analysis, among which is Scanning Electron Microscopy (SEM). Furthermore, phase contrast is widely used in nematology for a variety of purposes. This chapter endeavors to provide a comprehensive understanding of the microscope, measurements, DIC, and SEM techniques.

Molecular techniques are crucial for research on nematology, and the processing of samples is essential for their long-term storage prior to analysis. This chapter focuses on DNA extraction, various DNA markers, and polymerase change reaction (PCR). To ensure the preservation of nematodes for extended periods, DESS can be utilized. Moreover, the quality of extracted DNA is vital for proper PCR processing. This chapter offers an overview of nematode isolation and the different techniques for DNA extraction. The utilization of molecular markers presents numerous benefits when studying the genetic makeup of nematode populations. Among these markers, sequence characterized amplified region (SCAR) stands out for its exceptional value in identifying nematodes belonging to the Meloidogyne species. Amplified fragment length polymorphism (AFLP), on the other hand, is a technique that can be employed to analyze the genetic variability of nematodes. It is important to note that these markers are primarily used for plant-parasitic nematodes, which have a significant impact on the economy. This chapter focuses on the most commonly utilized marker for nematodes. After extracting DNA, the next step involves amplifying the target genes through PCR. Various primers, including rDNA and mtDNA, are available for nematode genes and serve useful taxonomic purposes. However, precise primer design is critical for achieving accurate nematode diagnosis. Both DNA and primers must be of high quality to ensure successful PCR. Eco-friendly standard dyes like SafeView can be employed to visualize PCR products. This chapter offers a comprehensive guide to PCR processing and DNA visualization techniques. 

Bioinformatics is the interdisciplinary study of nucleic acid and protein sequences, which has proven especially useful for genomics, gene expression, and nematode diagnosis. Quality control of these sequences is essential, and bioinformatics plays a crucial role in their processing for phylogenetic analysis. Skilled analysis is particularly vital for molecular ecology and environmental DNA analysis, especially when working with next-generation sequences. Furthermore, the utilization of an online database and specialized software designed for the identification of nematode species serves as valuable resources for a proper diagnosis of nematode-related issues. In this chapter, we will explore the bioinformatics of Sanger sequencing, with an emphasis on the phylogenetic study of nematodes and online species identification of nematode species. 

This chapter delves into the assessment of nematode biodiversity, which involves investigating the community of these organisms present in a given region. The biodiversity of nematodes can be studied through molecular or conventional approaches. In a conventional method, proper sampling is essential to accurately evaluate biodiversity, and this task is aided by the use of various indices, including Shannon, richness, evenness, and community indices. In the field of molecular biodiversity, researchers can investigate the diversity of nematodes by conducting metagenomic analysis of ribosomal DNA (e.g., 18S rDNA) or cytochrome c oxidase subunit I (cox1) gene of mitochondrial DNA. These approaches provide significant insights into the identification and classification of nematode taxa present in soil ecosystems. Analysis of these genetic markers allows scientists to better understand the rich diversity of nematodes and their ecological roles within soil communities. In this chapter, relevant information on nematode biodiversity assessment is presented.

 The maintenance of live nematodes is a crucial task for various studies related to their biology, taxonomy, and genetics. It is essential to have a living source of nematodes for future research purposes. Researchers typically use media or plants to culture and multiply nematodes. While agar serves as a base ingredient for several nematodes, liquid or solid culture media is used for laboratory rearing. It is important to note that culturing plant-parasitic nematodes is more challenging in the lab than free-living nematodes. However, entomopathogenic nematodes can be cultured in large quantities for pest control purposes. This chapter provides a thorough discussion of the culturing methods of several nematodes. Greenhouses are an essential tool for nematologists to study nematodes in the field of integrated pest management. By using various types of greenhouses, researchers can achieve their goals in nematode biology. The Phytotron provides the most precise and controlled environment for biological studies, while certain nematodes, such as Meloidogyne and cyst nematodes, can be mass-reared in greenhouses for further molecular or biological surveys. This chapter delves into the important tasks associated with greenhouses for economically significant plant-parasitic nematodes.