Summary 1.One of the ongoing challenges in molecular biology is to develop rapid, accurate and reliable techniques to identify organisms. This contribution evaluates the value of DNA melt peak analysis for the identification of pests and pathogens significant to biosecurity. 2. The method evaluated in this study capitalises on the observation that double-stranded DNA separates into single strands (melts) at a temperature dependant on the nucleotide sequence of the DNA strands. The temperature and shape of melt peaks from polymerase chain reaction (PCR) products using standard primers and amplification protocols were evaluated for species identification, and the advantages of this method over agarose gel electrophoresis exemplified. 3. Three insect (weevil) species were discriminated by the melt profile shape and temperature of their cytochrome oxidase subunit I (COI) PCR product using order-specific universal primers. 4. Three of four arachnid (tick) species were discriminated by the melt profile shape and temperature of their COI and 18S PCR product using order-specific universal primers. 5. Plum pox virus-specific primers amplified only the target virus that was verified by its unique melt temperature rather than by conventional staining of the amplicon on an electrophoretic gel. 6. Melt peak analysis is a useful and robust tool for the rapid detection of PCR products and the identification of organisms. An efficiency study revealed this technique required half the time input, and cost half as much, as PCR followed by AGE.

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Sitona lepidus had spread throughout the North Island of New Zealand by 2005 and was first detected in the South Island in January 2006 when one individual was found at Harewood Christchurch Intensive sampling during February 2006 recovered only two additional specimens Several specimens were recovered from a separate Christchurch location in August 2006 Localised S lepidus populations were discovered near Richmond Nelson in April 2006 and in Rai Valley in May 2006 A website established in May 2006 to provide information about S lepidus was visited a mean of 135 times per month but it was never used to report possible new South Island infestations A biological control agent Microctonus aethiopoides was released at Richmond and Rai Valley between August 2006 and March 2007 By May 2007 it was parasitising from 4 to 14 of S lepidus adults which indicates it is likely to become permanently established

Positive controls are essential for PCR reliability and are challenging to obtain for rare, exotic and/or emerging pathogens and pose biosafety risks if manufactured using infectious pathogens. Custom synthetic DNA inserts can be designed de novo in tandems of forward and reverse complement priming sequences to be inserted in circular plasmid vectors. To test this concept, artificial positive controls (APCs) for use in PCR were synthesized to contain primer sequences targeting four viruses (Barley yellow dwarf virus, Soilborne wheat mosaic virus, Triticum mosaic virus and Wheat streak mosaic virus) pathogenic to wheat and, as internal control, the plant mitochondrial nad5 gene. Thermodynamics and folding parameters of twenty-four APC inserts were assessed in silico. Two thermodynamically different APCs, designated optimal and sub-optimal, were cloned and tested using end point PCR. The optimal APC had a 100% amplification rate, while only 92% of virus-infected plant tissues, commonly used as reference positive controls, amplified. An array of APC priming sequences from different organisms and/or previously tested primers can be accommodated in a large and flexible number of positive control targets. APCs will streamline and standardize routine PCR, improve reliability and biosafety, and create opportunities for development and commercialization of new synthetic positive control sequences.

Eight South American geographical populations of the parasitoid Microctonus hyperodae Loan were collected in South America (Argentina, Brazil, Chile and Uruguay) and released in New Zealand for biological control of the weevil Listronotus bonariensis (Kuschel), a pest of pasture grasses and cereals. DNA sequencing (16S, COI, 28S, ITS1, beta-tubulin), RAPD, AFLP, microsatellite, SSCP and RFLP analyses were used to seek markers for discriminating between the South American populations. All of the South American populations were more homogeneous than expected. However, variation in microsatellites and 16S gene sequences corroborated morphological, allozyme and other phenotypic evidence of trans-Andes variation between the populations. The Chilean populations were the most genetically variable, while the variation present on the eastern side of the Andes mountains was a subset of that observed in Chile.