Summary A real‐time quantitative PCR technique has been used to develop a rapid and sensitive seed health test for Pyrenophora spp. on barley seed. Using the fluorescent reporter dye SYBR Green I for real‐time detection of PCR amplification, pathogen DNA extracted from infected seed can be quantified to the picogram level. The amount of Pyrenophora DNA extracted from seed samples of an artificial infection level gradient, constructed by mixing infected and uninfected seed, correlated with good agreement (r = 0.931) to percentage infection levels of the same samples measured by agar plate testing. In addition, a correlation of r = 0.883 was obtained between the two testing methods for naturally infected seed, ranging from 0% to 89% infection. Samples could be quantified to below the 2% voluntary threshold required for deciding on seed treatment. The proposed test was performed in three parts: (i) quantification of Pyrenophora spp. infection using Pyrenophora‐specific PCR primers; (ii) test of any negative samples from (i) with barley‐specific PCR primers to check the DNA extraction process; (iii) test of positive samples from (i) for the presence of Pyrenophora graminea using P. graminea‐specific PCR primers. All PCRs were performed in the LightCycler™ instrument allowing each PCR run and analysis to be completed within 30 min. With the current daily receipt of samples (batches up to 16) the test can be completed in 8 h, compared to 7 days for the traditional agar plate test.

Sixty RAPD primers were used to screen for a diagnostic marker that could be used to identify Pyrenophora graminea, a fungal seedborne pathogen that causes leaf stripe on barley. Primer pairs were designed to differentiate P. graminea from other Pyrenophora spp. using a sequence‐characterized amplified region (SCAR) approach. A pair of P. graminea‐specific primers (PG2 F/R) was obtained that amplified a single fragment from 37 isolates of P. graminea tested, but not from 29 isolates of other Pyrenophora spp. or 12 saprophytes isolated from barley seed. Rapid PCR detection was achieved using a LightCycler, in which the emission of fluorescence from the binding of SYBR Green I dye to the PCR products is measured. The P. graminea‐specific product resulting from amplification with PG2 F/R can be distinguished from any nonspecific products by post‐PCR melting point analysis. The PCR assay involves 40 amplification cycles of PCR, and the total PCR test including melting point analysis takes 25 min to complete. The rapidity of this test, combined with the closed ‘in‐tube’ detection of PCR products, which reduces the potential for contamination, offers significant advantages compared with conventional laboratory and PCR analyses.

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The phloem limited bacterium ‘Candidatus Liberibacter solanacearum’ (Lso) is associated with disease in Solanaceous and Apiaceous crops. This bacterium has previously been found in the UK in Trioza anthrisci, but its impact on UK crops is unknown. Psyllid and Lso diversity and distribution among fields across the major carrot growing areas of Scotland were assessed using real-time PCR and DNA barcoding techniques. Four Lso haplotypes were found: C, U, and two novel haplotypes. Lso haplotype C was also found in a small percentage of asymptomatic carrot plants (9.34%, n = 139) from a field in Milnathort where known vectors of this haplotype were not found. This is the first report of Lso in cultivated carrot growing in the UK and raises concern for the carrot and potato growing industry regarding the potential spread of new and existing Lso haplotypes into crops. Trioza anthrisci was found present only in sites in Elgin, Moray with 100% of individuals harbouring Lso haplotype C. Lso haplotype U was found at all sites infecting Trioza urticae and at some sites infecting Urtica dioica with 77.55% and 24.37% average infection, respectively. The two novel haplotypes were found in Craspedolepta nebulosa and Craspedolepta subpunctata and named Cras1 and Cras2. This is the first report of Lso in psyllids from the Aphalaridae. These new haplotypes were most closely related to Lso haplotype H recently found in carrot and parsnip. Lso was also detected in several weed plants surrounding carrot and parsnip fields. These included two Apiaceous species Aegropodium podagraria (hap undetermined) and Anthriscus sylvestris (hap C); one Gallium sp. (Rubiaceae) (hap undetermined); and Chenopodium album (Amaranthaceae) (hap undetermined).

The overall aim of this study was to develop a new, reliable and rapid diagnostic assay for differentiating six European Armillaria species based on variation in their elongation factor-1 alpha (EF-1 a) gene sequences and to verify a set of species-specific primers on 61 Armillaria isolates from Europe. Partial sequences of the EF-1 a gene obtained in Armillaria borealis, Armillaria cepistipes, Armillaria gallica, Armillaria mellea, Armillaria ostoyae and Armillaria tabescens revealed sufficient interspecific variation to distinguish among species using nested primers. These primers gave unambiguous bands when tested on representative isolates of five of these species. However, the EF-1 a sequences of European A. borealis isolates clustered into two distinct clades, termed here AbX and AbY. Specific primers were subsequently designed and tested successfully on both AbX-type and AbY-type A. borealis isolates. The taxonomy of A. borealis needs to be elucidated to determine whether a new, as yet unnamed Armillaria taxon exists in Europe. Three A. borealis isolates were also found to have heterozygous sites in their EF-1 a sequences, which suggests that the gene could exist in more than one copy or that these isolates contain hybrid sequences. A pyrosequencing method was also developed, targeting a small region of EF-1 a intron 4, which was able to differentiate European Armillaria isolates to the species level and additionally could distinguish AbX-type and AbY-type A. borealis isolates.