METHODS AND TECHNIQUES FOR NEMATOLOGY

1 METHODS AND TECHNIQUES FOR NEMATOLOGY By J. van Bezooijen Revised version 2006 PREFACE This Manual METHODS and TECHNIQUES for NEMATOLOGY is based on the Manual for Practical Work in NEMATOLOGY (s Jacob & van Bezooijen 1984), which has been thoroughly revised. The Practicumhandleiding Nematologie (van Bezooijen & Ettema 1996) was the basis for even further revision. The final product, which you will be using now, does not pretend to be complete, but is an augmented and in many parts corrected version of the earlier manuals.

2 The author is greatly indebted to A. Raeymaekers for preparing the final layout and for her useful proposals to increase the readability. Her impressive computer skills contributed substantially to a higher quality of the book. Additional improvements and corrections may remain necessary and the author welcomes any comments and suggestions. In spite of this, hopefully this manual will be useful to you Wageningen, December 2006 CONTENT PREFACE CONTENT CHAPTER 1. SAMPLING 2 PRACTICAL AND THEORETICAL CONSIDERATIONS 2 Purpose and accuracy of sampling 2 Variation in space: horizontal and vertical 2 Variation in time.

3 Seasonal fluctuations and life cycles 3 Statistics 3 THE SAMPLING PROCESS 4 Tools 4 Sampling patterns 5 Examples of sampling METHODS 5 TRANSPORT AND STORAGE 8 Fixing samples in the field

4 8 PRE-PROCESSING OF THE SAMPLE 8 Measuring: volume or weight? 8 Taking sub-samples; Mixing 9 Pre soaking of soil samples 10 Drying 10 CHAPTER 2.

5 EXTRACTION 11 INTRODUCTION 11 Principles 11 Extraction from plant material 11 Extraction from soil and other substrates 12 Extraction of cysts 12 Selection of an extraction method 14 EXTRACTION

6 FROM PLANT MATERIAL 18 Dissection 18 Baermann funnel 19 Funnel spray method 21 Blender nematode filter method 23 Blender centrifugal flotation method 25 EXTRACTION FROM SOIL AND OTHER SUBSTRATES 27 Nematode cotton wool filter method 27 Decanting and sieving.

7 Cobb s method 30 Erlenmeyer- or (milk) bottle method 34 Oostenbrink elutriator 37 Standard use 38 METHODS and TECHNIQUES for NEMATOLOGY Modification for the extraction of large nematodes 42 Modification for the extraction of stem nematodes 43 Centrifugal flotation

8 Method 46 Modification for the extraction of fixed samples 51 EXTRACTION OF CYSTS 52 Baunacke method 52 Fenwick can 54 Kort s cyst extraction elutriator 56 Seinhorst cyst extraction elutriator 58 Isolating cysts from the debris 60 Isolating cysts with organic solvents 60 Isolating cysts with

9 The centrifugal flotation method 62 ANALYSIS OF EXTRACTS 64 Counting of nematodes in the suspension 64 Estimating and counting the cyst content 65 Presentation of numbers 66 CHAPTER 3. PROCESSING 68 PICKING NEMATODES 68 CONCENTRATING NEMATODE SUSPENSIONS 68 ANAESTHETIZING