An in Vitro Technique for Distinguishing Between Live and Dead Meloidogyne Incognita Juveniles

Meloidogyne incognita (Root-knot Nematode, RKN) is one of the most important plant-parasitic nematodes which causes great damage to cotton in the U.S. The objective of this research project was to develop a quick method to distinguish between live and dead second stage juveniles (J2’s) of M. incognita, which is the key point for any in vitro screening of chemical or biological control agents on paralysis of RKN J2’s. The best irritant was selected from 1N sodium carbonate (Na₂CO₃), 1N sodium bicarbonate (NaHCO₃), and 1N sodium hydroxide (NaOH) at pH=10. In vitro 96 well plates were used and 30-40 RKN J2’s were pipetted in each well. 1 µl, 10 µl, and 20 µl of each sodium solutions were tested to find the best concentration. Numbers of live J2’s were counted and recorded before exposure and at 30 minutes after the addition of the sodium treatment. Each treatment had four replications and each trial was repeated twice. Data collected were analyzed in SAS 9.2 (SAS Institute, Inc.) by using PROC Glimmix procedure and Dunnett's t-test with P ≤ 0.05. Results indicated that 1 µl 1N NaOH at pH=10 effectively (P ≤ 0.05) determined live RKN J2’s. The 1 µl 1N NaOH also caused the nematodes to display a rapid twisting movement and curling after a two minutes exposure which was visibly distinguishable. Death J2’s were straight and didn’t move. In conclusion, 1 µl 1N NaOH with 100 µl of sterile water is the optimum sodium agitation for rapidly distinguishing live RKN J2’s from the dead ones. Further growth chamber test will be done to confirm that live J2’s are really alive and infective. The technique will be applied to high throughput in vitro screening of chemical or biological control agents on RKN J2’s.