The added value of proficiency tests: choosing the proper method for extracting Meloidogyne second-stage juveniles from soil

Abstract

To gain insight into the different extraction processes used in laboratories in various European countries and the effect these methods might have on the number oi Meloidogyne second-stage juveniles (J2) extracted from soil samples, a ring test with 2 elements was conducted. The first element used the Baermann funnel as a method performance test. The second element was a proficiency test, in which the laboratory's standard technique was compared with the Baermann method. Chilled samples of 100 cm^ (10 per extraction method) were sent to participants in insulated containers. In the nematode counts distinction was made between Meloidogyne J2, other plant-parasitic nematodes and/or saprophytes. A total number of 18 participants took part in the method performance test and 13 institutes took part in the proñciency test using 11 different extraction techniques. The initial population density in the soil was 2025 J2 (100 cm^ soil)"', detemnined by Oostenbrink elutriator and 4 weeks incubation. In the method performance test the institutes found Meloidogyne J2 numbers varied between 0 and 705 J2 (100 cm^)-^ Using the Baermann extraction method the median number per institute ranged from 0 to 377 J2 (100 cm^)~'. In the proficiency test comparison amongst the different methods showed an increase of more than 100-fold, with the highest median of 3733 J2 (100 cm^)"' and the lowest of 34 J2 (100 cm^)"^ the difference was caused mainly by the incubation effect. For the other plant-parasitic nematodes and the saprophytes, the differences between the most and least efficient extraction methods were much smaller. The variance of the Meloidogyne counts was the highest for the standard Baermann extraction technique; other similar techniques also had high variances. The automated zonal centrifuge had the lowest variance. The Baermann method is not advisable for survey purposes.

PM 7/119 (1) Nematode extraction

“…Over past years, many methods have undergone test performance studies nationally and internationally, but only few results have been published (e.g. Müller, ; Ladeveze & Anthoine, ; den Nijs & van den Berg, ). Nevertheless, it became evident from these tests that results between laboratories can vary greatly.…”

Development of a direct quantitative detection method for Meloidogyne incognita and M. hapla in andosol and analysis of relationship between the initial population of Meloidogyne spp. and yield of eggplant in an andosol

“…Indeed, the Baermann method extracted only 30% of plant-parasitic nematodes recovered by the double-layer centrifugation method which extracts passive nematodes in soil as well as active nematodes (McSorley and Frederick, 2004). In addition, Den Nijs and Van Den Berg (2013) revealed that the variance of Meloidogyne counts was the highest for the Baermann method, and concluded that the Baermann method is not advisable for survey purposes. An alternative quantification method with real-time PCR has been developed that enables quantifying all forms of nematodes (Goto et al, 2009;Min et al, 2011).…”