Abstract:
Root-knot nematodes (RKNs) from the genus Meloidogyne are major obligate
endoparasitic pathogens of a multitude of economically important host plants worldwide.
Correct identification of RKN species is becoming increasingly important for the design of
effective nematode management practices. To our knowledge there were no literature
records about molecular identification of RKNs in Sri Lanka. The main objective of this
study was to develop a fast, accurate and sensitive Polymerase Chain Reaction (PCR)
based molecular assay to accurately identify RKNs parasitizing important vegetable and
fruit crops in Sri Lanka. Root knot nematode infected root samples were collected from 4
different localities in Sri Lanka during the years 2013-2014 and labeled as 1-
Horana/Tomato, 2- Horana/Spinach, 3-Anuradhapura/Guava, 4-Kalpitiya/Tomato, 5-
Colombo/Cherry Tomato, 6- Kalptiya/ Guava. DNA extracted from juvenile stages of
Meloidogyne was used in PCR. Amplification of ribosomal DNA with MF/MR universal
primers yielded a 500 bp fragment specific for genus Meloidogyne for samples belonging
to all localities. The sequence of 500 bp PCR product was found to be 100 % identical to
the most common Meloidogyne species available in Genbank. C2F3/1108 primers that
result in species-specific PCR products based on size were used to identify and
differentiate infected Meloidogyne species in the collected samples. Amplification of
mitochondrial DNA with C2F3/1108 primers yielded an 1100 bp product specific for M.
arenaria for only sample 1, a 705 bp size products specific for M. enterolobii for sample 1,
3 and 6, a 520 bp products specific for M. chitwoodi or M. hapla for sample 2, 4, 5 and 6.
Sample 2, 4, 5 and 6 were further analyzed with 194/195 primers and the exact RKN
infected was identified as M. hapla by the amplification of specific ~ 700 bp size PCR
product. This study demonstrated the occurrence of Meloidogyne species in Sri Lanka
either alone or in mixed populations. Even though the species M. arenaria, M. incognita, M.
javanica, and M. hapla are generally considered the most widespread, M. enterolobii and
M. hapla were found to be more widely distributed in the studied areas of Sri Lanka. The
protocols optimized in this study would be useful in the future to analyze RKN infected
samples collected across Sri Lanka to evaluate the prevalence, incidence and diversity of
RKNs more comprehensively.
