Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

Abstract

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii). Key words: clover cyst nematode, corn cyst nematode, diagnosis, Heterodera glycines, Heterodera goettingiana, Heterodera schachtii, Heterodera trifolii, Heterodera zeae, molecular nematology, nematode PCR-RFLP, pea cyst nematode, rDNA variation, regulatory nematology, soybean cyst nematode, sugar beet cyst nematode.