Phylogenetic analysis of New Zealand tomato spotted wilt virus isolates suggests likely incursion history scenarios and mechanisms for population evolution.

Tomato spotted wilt virus (TSWV) is an internationally significant pathogen with a wide host range, vectored by thrips. We have studied the sequence variation and evolutionary mechanisms at play in parts of the L, M and S subgenomes of 23 New Zealand TSWV isolates collected between 1992 and 2009, aiming to identify the possible geographic origins of isolates. Maximum-likelihood-based phylogenetic analyses of New Zealand and overseas TSWV isolates placed the L and M subgenome sequences of two isolates (MAF04 and PFR04) in distinct clades composed primarily of Korean, Japanese and Chinese isolates, in contrast to the remaining 21 isolates, which clustered with a cosmopolitan group of isolates. The nucleocapsid (N) gene sequences of MAF04 and PFR04 plus MAF02 clustered with Japanese isolates. Consequently, we postulate that these isolates may represent a distinct incursion into New Zealand, but we do not have enough evidence to indicate an incursion pathway. Alternately, these isolates may have arrived with an incursion that included a mixture of TSWV isolates of diverse international origins. The sequences of four of the TSWV isolates contained a number of sites with a mixture of nucleotides, suggesting that these isolates either consisted of several sequence variants or were from plants with mixed infections. One isolate (MAF02) was shown to be a either a reassortant or an S subgenome recombinant. Large amounts of low-level polymorphism were detected with low amino acid change fixation rates (purifying selection). Negative selection was indicated at four amino acid sites in the New Zealand TSWV N gene sequences.

Genomics of biotrophic, plant-infecting plasmodiophorids using in vitro dual cultures.

The plasmodiophorids are a phylogenetically distinct group of parasitic protists that infect plants and stramenopiles, causing several important agricultural diseases. Because of the obligate intracellular part of their lifecycle, none of the plasmodiophorids has been axenically cultured. Further, the molecular biology of the plasmodiophorids is poorly understood because pure cultures are not available from any species. We report on an in-vitro dual culture system of the plasmodiophorids Plasmodiophora brassicae and Spongospora subterranea with their respective plant hosts, Brassica rapa and Solanum tuberosum. We show that these plasmodiophorids are capable of initiating and maintaining stable, long-term plant cell callus cultures in the absence of exogenous plant growth regulators. We show that callus cultures harbouring S. subterranea provide an excellent starting material for gene discovery from this organism by constructing a pilot-scale DNA library. Bioinformatic analysis of the sequences established that almost all of the DNA clones from this library were from S. subterranea rather than the plant host. The Spongospora genome was found to be rich in retrotransposable elements, and Spongospora protein-coding genes were shown to contain introns. The sequence of a near full-length non-LTR retrotransposon was obtained, the first transposable element reported from a cercozoan protist.