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

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

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

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

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 http://www.ctahr.hawaii.edu/noni/gallery/1_noni_root_knot_galls_on_young_roots1.JPG Introduction Materials and Methods Results Conclusions

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

Experimental Design Introduction Materials and Methods Results Conclusions

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

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

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

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

Introduction Materials and Methods Results Conclusions

Frequency of Fatty Acids Introduction Materials and Methods Results Conclusions

Combined Profiles

Significant Peaks 1st Canonical Variate 2nd Canonical Variate

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 < 0.0001 D2 = 26; P < 0.0001 Introduction Materials and Methods Results Conclusions

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 < 0.0001 Introduction Materials and Methods Results Conclusions

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

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

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

References Gazaway, W. S., and K. S. McLean. 2003. 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. 1973 A comparison of methods of collecting inocula of Meloidogyne spp. Including a new technique. Plant Disease Reporter 57:1025-1028 Jenkins, W. R. 1964. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Disease Reporter 48:692 Sasser, M. 1990. Identification of bacteria through fatty acid analysis. Methods in phytobacteriology pp. 199-204

Acknowledgements Plant Science Research Center, Auburn AL