Investigating the genetic diversity of Puccinia boroniae in Western Australia
Driessen, S.A., O'Brien, P.A. and Hardy, G.E.St.J. (2006) Investigating the genetic diversity of Puccinia boroniae in Western Australia. In: 8th International Mycological Congress, 21 - 25 August, Cairns, Queensland.
Puccinia boroniae is an economically important pathogen in commercial Boronia plantations in Western Australia. To address the question of whether one or more species of P. boroniae are present an initial study was conducted. Genetic diversity among field specimens collected from commercial Boronia plantations was assessed by analysis of the polymorphism within the intergenic spacer 2 (IGS2) region of the nuclear ribosomal RNA genes.
The IGS2 region of 22 P. boroniae specimens was amplified with primers CNS1 and NP. PCR products were subsequently digested with a selection of restriction enzymes and analysed by gel electrophoresis. Five representative samples were purified, cloned into pCII-TOPO vector, the cloned fragments sequenced and aligned.
Two different RFLP profiles were generated. Most specimens, sampled from a range of Boronia species and collected from plantations at different geographical locations, produced a homologous RFLP profile (Group 1). Three specimens (Group 2), obtained over three sampling periods from Boronia megastigma at a single plantation, produced a different profile. A single specimen also collected from this same plantation, but isolated from B. heterophylla belonged to Group 1.
Comparison of sequence data generated from representative specimens from each profile group showed that single point mutations at endonuclease recognition sites were responsible for the changes in profile. Sequence alignment also highlighted several insertion/deletion events common to the Group 2 specimens.
Variation between single telia of P. boroniae collected from individual B. megastigma plants at the plantation exhibiting the Group 2 profile was examined by PCR-RFLP analysis of the nuclear internal transcribed spacer (ITS) region. A single homologous profile was observed.
Overall, no segregation of P. boroniae according to host specificity was concluded, though the data suggested the possible presence of a subspecies (race) of P. boroniae, that is isolated geographically and possibly host [cultivar) specific. Further investigations involving pathogenicity trials are required. The low level of diversity observed in this study may also be a reflection of the influence of human distribution on the pathogen, as the rust is rarely observed in wild populations .
An interesting aspect of the study was the presence of a new spore stage (pycnial stage) recorded only at the plantation at which the Group 2 specimens were sampled from. The life cycle role of this spore stage is unconfirmed at present.
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