Differentiation of three Bursaphelenchus species by RAPD-PCR
Studies have been carried out in Germany on the use of RAPD-PCR to differentiate Bursaphelenchus xylophilus (EPPO A1 quarantine pest) from two other related species, B. mucronatus and B. fraudulentus. These species present close morphological similarities. Species identification is based mainly on tail features of the females. B. xylophilus is normally round-tailed, whereas B. mucronatus and B. fraudulentus have a more or less pointed (mucronate) tail tip. However, some North American B. xylophilus has both round-tailed (r-forms) and mucronate forms (m-forms). Diagnostic techniques based on nucleic acid sequence are more reliable than microscopy and enzyme electrophoresis. Therefore RAPD-PCR has been used on 14 isolates of B. xylophilus (r-forms from CN, JP, US and CA, and m-forms from US, CA), 12 isolates of B. mucronatus (from CA, FR, RU, DE, NO, FI, IT, JP, CN), 7 isolates of B. fraudulentus (from AT, HU, DE) and 2 undefined Bursaphelenchus (from US). 13 different oligonucleotide primers were used and most primers showed interspecific and intraspecific differences between isolates; 3 primers were retained as giving the most satisfactory results in consistently differentiating species. The results obtained showed that with two RAPD-PCR tests (using two primers) it is possible to differentiate B. xylophilus from B. mucronatus and B. fraudulentus. Until now, North American isolates were considered as being only B. xylophilus, but these studies showed that a Canadian isolate could clearly be reclassified as B. mucronatus (this confirms also earlier studies on this isolate). The two previously undefined Bursphelenchus isolates from USA were shown to be B. fraudulentus demonstrating, for the first time, the presence of this species in North America. Surprisingly, these isolates came from conifers (whereas in Europe B. fraudulentus occurs on deciduous trees). The authors wondered whether B. fraudulentus has a different mode of life in America or relies on a vector beetle attacking both coniferous and deciduous trees. These studies revealed considerable genetic differences between populations of B. mucronatus; in particular Asian provenances showed very unique patterns. The authors suggest that a subspecies status for European, Siberian and North American provenances of B. mucronatus should be considered (as the results of crossing experiments do not justify a separate species status). RAPD patterns have also revealed considerable intraspecific heterogeneity within B. xylophilus. The tested primers did not differentiate between round-tailed and mucronated populations of ;B. xylophilus. The authors conclude that further studies will be necessary on intraspecific differentiation of the various provenances of B. xylophilus and B. mucronatus and that the possibility of naturally occurring hybrid populations should also be considered.
Sources
Braasch, H.; Burgermester, W.; K.-H. Pastrik (1995) Differentiation of three Bursaphelenchus species by means of RAPD-PCR.
Nachrichtenblatt des Deutschen Pflanzenschutzdienstes, 74, 310-315.