1. Fingerprinting Nematode Fatty Acid Compositions as a Means for Identification
N. S. Sekora, K. S. Lawrence, J. A. McInroy
Department of Entomology and Plant Pathology
Auburn University, Auburn AL

2. Purpose
To identify nematode species without using traditional microscopic techniques by analysis of fatty acids
Introduction Materials and Methods Results Conclusions

3. Hypothesis Fatty acid profiles of nematodes should vary between genera
Profiles should vary enough to differentiate between genera
Introduction Materials and Methods Results Conclusions

4. Rotylenchulus reniformis
Reniform nematode
First reported in 1940
Now most costly cotton pathogen in Alabama
Gazaway and McLean, 2003
Infects by embedding anterior end into root tissue
100x
Introduction Materials and Methods Results Conclusions

5. Meloidogyne incognita
100x
Pathogen of cotton, peanuts, and corn
Smallest of plant- parasitic nematodes
Embeds entire body
Forms gall on root tissues
Stunts plant growth
Introduction Materials and Methods Results Conclusions

6. Heterodera glycines Soybean cyst nematode
Largest plant-parasitic nematode
Female forms cyst that contains eggs
25x
Introduction Materials and Methods Results Conclusions

7. Experimental Design
Introduction Materials and Methods Results Conclusions

8. Isolation of Nematode Samples
Nematodes extracted from greenhouse samples
Juvenile forms extracted by sucrose-floatation
Jenkins, 1964
Eggs extracted via 0.6% NaOCl
Hussey and Barker, 1973
Samples enumerated for dilutions
Introduction Materials and Methods Results Conclusions

9. Isolation of Nematode Samples
Samples containing 1–100 nematodes hand-picked
Larger samples by dilution
Samples can be frozen for later extraction
Introduction Materials and Methods Results Conclusions

10. Extraction of Fatty Acids
Extracted as per procedure described by Sasser, 1990
Soaponification
Methylation
Extraction
Base wash
Additional step
Samples evaporated and reconstituted in 150μL of the organic extraction solvent
Concentrates samples
Introduction Materials and Methods Results Conclusions

11. FAME Analysis Automated analysis via gas chromatography
Fatty acids listed by time to pass through column
Larger and more modified slower
Concentrations listed by percentage present
Introduction Materials and Methods Results Conclusions

12. Introduction Materials and Methods Results Conclusions

13. Frequency of Fatty Acids
Introduction Materials and Methods Results Conclusions

14. Combined Profiles

15. Significant Peaks
1st Canonical Variate
2nd Canonical Variate

16. Canonical Analysis
Fatty acid 20:4 ω6,9,12,15c is perfectly correlated along the 1st canonical variate
Other fatty acids contribute
18:1 ω5c
18:2 ω6,9c
15:1 anteiso A
12:0 2OH
D2 = 32; P <
D2 = 26; P <
Introduction Materials and Methods Results Conclusions

17. Canonical Analysis
The fatty acids 15:0 iso and 18:0 3OH nearly perfectly correlate along the 2nd canonical variate
The remaining four fatty acids further separate the genera
16:1 ω5c
18:1 ω9c
14:0
17:0 iso
D2 = 3.8; P <
Introduction Materials and Methods Results Conclusions

18. Conclusions
Fatty acids can be used as a means to differentiate between genera of nematodes
Certain fatty acids are unique in their expression among nematode genera
Introduction Materials and Methods Results Conclusions

19. Possible Applications
Identify nematode species in soil samples without using microscopy
Build a library of profiles for recognition of genera with the Sherlock Analysis Software
Introduction Materials and Methods Results Conclusions

20. Future Studies Determine the fatty acid profiles of other genera
Evaluate the possibility of species identification within genera
Identify concentrations of multiple nematodes in mixed-genera samples
Identify nematode species within soil extractions
Determine the effect of hosts on fatty acid profiles
Introduction Materials and Methods Results Conclusions

21. References
Gazaway, W. S., and K. S. McLean A survey of plant parasitic nematodes associated with cotton in Alabama. Journal of Cotton Science 7:1-7.
Hussey, R. S., and K. R. Barker A comparison of methods of collecting inocula of Meloidogyne spp. Including a new technique. Plant Disease Reporter 57:
Jenkins, W. R A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Disease Reporter 48:692
Sasser, M Identification of bacteria through fatty acid analysis. Methods in phytobacteriology pp

22. Acknowledgements
Plant Science Research Center, Auburn AL