Techniques for Work with Plant and Soil Nematodes

Roland N Perry, David Hunt, Sergei A Subbotin, Roland N Perry, David J Hunt, Sergei A Subbotin

• Publisher: CAB International
• Year: 2020
• Print ISBN: 9781786391759
• eBook ISBN: 9781786391773
• Topic: Biological Sciences
• Subtopic: Botany
• Index: Biological Sciences

Plant-parasitic and free-living nematodes are increasingly important in relation to food security, quarantine measures, ecology (including pollution studies), and research on host-parasite interactions. Being mostly microscopic, nematodes are challenging organisms for research. Techniques for Work with Plant and Soil Nematodes introduces the basic techniques for laboratory and field work with plant-parasitic and free-living soil-dwelling nematodes. Written by an international team of experts, this book is extensively illustrated, and addresses both fundamental traditional techniques and new methodologies. The book covers areas that have become more widespread over recent years, such as techniques used in diagnostic laboratories, including computerized methods to count and identify nematodes. Information on physiological assays, electron microscopy techniques and basic information on current molecular methodologies and their various applications is also included.This book is an essential resource for students of nematology and parasitology, academic researchers, diagnostic laboratories, and quarantine and advisory service personnel. It provides a much-needed methodology standard for anyone involved in work on plant and soil nematodes.

1 Sampling

LOES J.M.F. DEN NIJS¹, JON PICKUP² AND RALF-UDO EHLERS³

¹National Reference Centre, Wageningen, The Netherlands; ²SASA, Edinburgh, UK; ³e-nema GmbH, Schwentinental, Germany

1.1 Introduction: The Purpose of Sampling

1.2 Sampling Strategies

1.3 Sampling in Relation to Phytosanitary Requirements

1.4 Soil Sampling for Endoparasitic Nematodes

1.5 Soil Sampling for Ectoparasitic Nematodes

1.6 Soil Sampling for Entomopathogenic Nematodes: Isolation and Baiting Techniques

1.7 Examples of Sampling Protocols for Pine Wood Nematodes and Potato Cyst Nematodes

1.8 Sampling Tools

1.9 Handling and Storage of Samples

1.10 References

1.1 Introduction: The Purpose of Sampling

The main drive for sampling is to know what is in there, ‘there’ being the matrix that one is interested in. This can vary from water, soil, wood, turf or bark, to any part of plants (seeds, stems, leaves, bulbs, etc.), as long as it concerns the plant-parasitic nematodes. When animal-parasitic nematodes are involved, the matrix will be parts of the infected animal, and when we look into entomopathogenic nematodes, soil and the parasitized insects will be the point of attention for sampling.

Nematodes can be present in various parts of plants or at various depths in the soil, depending on the circumstances and the life stage. This means that one should be aware of these possibilities when collecting samples. When soil is too wet, it is better to wait until the soil has reduced to field capacity. The same is true for when the soil is too dry. Nematodes need water and in dry conditions the nematodes will move to deeper soil layers. In general, soil samples can best be taken when the soil is moist. Some life stages are immobile and can only be found in the plant roots, whereas other stages can be found in the soil. This means that samples should be taken from the proper matrix at the appropriate time, taking into account the developmental stages the nematodes might be in. All the above-mentioned aspects should be kept in mind when sampling for nematodes.

In this chapter, the purpose of sampling, sampling techniques and, related to this, the sampling tools, and the handling and storage of the samples before processing of plant-parasitic and entomopathogenic nematodes will be discussed. Some protocols will be described in detail as examples.

1.2 Sampling Strategies

1.2.1 Diagnostic sampling

Plant-parasitic nematodes are generally not visible to the naked eye and the symptoms they produce are often attributed to fungi, viruses, bacteria, other pests or poor soil conditions, including a deficiency or excess of nutrients. As nematode damage can easily be overlooked, it is very important to sample plants and/or soil for nematodes when growth is unexpectedly poor or when symptoms cannot clearly be attributed to other causes. This type of sampling is called diagnostic sampling, as the reasons for poor growth of plants or trees need to be determined. In these situations a sample from the poorly growing plant/tree should be compared with a sample from a healthy one. When possible, a soil, leaf or root sample from the middle of the poorly growing patch of plants should be taken. For poorly growing trees samples from roots, leaves/needles or borings from trunks may be necessary. For comparison, a sample should also be taken from a healthy-looking plant/tree and/or the soil beneath it. In addition, when the patch of poor growth is clearly visible, it is wise to take a soil sample from the transition area (the area between healthy and poorly growing plants). Where plants are dead, no sample should be taken from that area or the dead plants as the nematode population is likely to have decreased markedly under these circumstances. The sample should then be taken from the less vigorous plants. In all situations, the nematodes have to be extracted from the plants/trees and soil using the appropriate technique: nematodes can be found in different parts of plants such as the roots, leaves and growing tips, and each matrix needs its own extraction method to separate the nematodes from the tissue or soil (see Viaene et al., Chapter 2, this volume). If possible, roots should either be included in the sample or taken separately; about 25–100 g, taken at random, should be sufficient, the lower weight being suitable for vegetables or citrus, whilst the higher weight being more applicable to plants with large roots such as banana. If stems and/or leaves appear to be attacked by nematodes, affected material can be removed and placed in polythene bags. Such samples should be kept separate from soil and/or root samples. To avoid misinterpretation, comparison of the nematodes found in the different situations can help determine whether nematodes in general or specific species/genera are involved.

In summary:

• Determine the poorly growing patch of plants/trees based on symptoms.

• Sample the soil from the centre of the poorly growing patch (but not under dead plants) using an auger or coring device, small trowel or a narrow-bladed shovel. A minimum of 500 g of soil should be taken.

• Take a complete plant sample from the centre of the patch of poor growth (but not a dead plant/tree).

• Repeat the above for the seemingly healthy situation and for the transition area when this is visible.

• Make sure that each sample is put into a (polyethylene) bag with label attached or inside and fill in all the necessary information to trace the sample back to its origin, e.g. sampling date, location (GPS coordinates), crop and cultivar plant species, name of sample taker, name of owner of crop/farmer, a reference number (when more samples are taken on one site). If possible, include details of the previous crop, soil type, treatments and other relevant information.

• If it is suspected that the pine wood nematode, Bursaphelenchus xylophilus, might play a role, wood from the trunk should be collected. See Section 1.7 for more detail and the sampling protocol.

1.2.2 Sampling for detection

When sampling for detection the question is usually ‘Are nematodes present in the field?’. When the objective is to detect the nematode in situations where the nematodes should not be present (see Section 1.3), sampling is similar to that for density estimates but the intensity of the sampling will be greater, related to the required detection level and the known distribution of the nematodes in the sampling unit. Sampling units might be soil samples, bulbs, plants or part of plants such as roots or growth tip.

Depending on the nematode groups likely to be responsible for the damage, the sampling depth may be very important. Nematodes are very mobile and avoid dry conditions. For virus vector nematodes, such as the genera Longidorus, Xiphinema and Trichodorus, Brown and Boag (1997) described the vertical distribution of these virus vectors as varying from 0 to 180 cm in depth. As these nematodes can be found deep in the soil, an auger of at least 40 cm depth should be used to collect soil samples. When the soil is too dry, in sub-tropical and tropical areas or in summer in temperate regions, the nematodes will have moved to deeper soil layers (even up to 100 cm depth) and soil sampling will become very difficult using a standard auger. In these situations a trowel can be used to remove the dry soil until the moist soil is reached and then take a soil sample. In general, a soil sample taken at ploughing depth should be sufficient in most cases in annual crops. In perennial and tree crops, soil sampling should take place as near to the root system as possible as Padusaini et al. (2006) already showed that the presence of the root system is the determinant factor in the vertical distribution of the nematodes.

Much current guidance suggests that when sampling for sedentary nematodes, such as cyst nematodes, cores should be taken to a depth of 15 cm, but there is little evidence to support this. Research by Been and ...

Table of contents

• Cover
• Dedication
• Half Title
• Title
• Copyright
• Contents
• About the Editors
• Contributors
• Preface
• 1 Sampling
• 2 Methods for Nematode Extraction
• 3 Estimating Numbers
• 4 Screening Plants for Resistance/Susceptibility to Plant-parasitic Nematodes
• 5 Handling, Fixing, Staining and Mounting Nematodes
• 6 Culturing Techniques
• 7 Measuring Nematodes and Preparation of Figures
• 8 Electron Microscopy Techniques
• 9 Behavioural and Physiological Assays
• 10 Staining Chromosomes
• 11 Isoelectric Focusing of Proteins
• 12 Molecular Identification of Nematodes using Polymerase Chain Reaction (PCR)
• 13 Isolation and Characterization of Tandem Repeats in Nematode Genomes
• 14 Characterization of Nematode Mitochondrial Genomes
• 15 Phylogenetic Analysis of DNA Sequence Data
• Index
• Cabi
• Back