Australian Journal of Entomology (2004) 43, 366–373 Blackwell Science, LtdOxford, UKAENAustralian Journal of Entomology1326-67562004 Australian Entomological SocietyMay 2004434366373Original ArticlePhytoplasma vector status of leafhoppersL J Pilkington et al. *Author to whom correspondence should be addressed (email: leigh.pilkington@ucr.edu) Vector status of three leafhopper species for Australian lucerne yellows phytoplasma Leigh J Pilkington, 1 * Geoff M Gurr, 1 Murray J Fletcher, 2 Alex Nikandrow 2 and Eric Elliott 3 1 Pest Biology and Management Group, The University of Sydney, PO Box 883, Orange, NSW 2800, Australia. 2 NSW Agriculture, Orange Agricultural Institute, Forest Road, Orange, NSW 2800, Australia. 3 NSW Agriculture, PO Box 369, Forbes, NSW 2871, Australia. Abstract The leafhoppers Orosius argentatus (Evans), Austroagallia torrida (Evans) and Batracomorphus angustatus (Osborn) were used in transmission tests to determine their vector status for the phyto- plasma associated with Australian lucerne yellows (ALuY). Caged, seed-grown lucerne plants were monitored for foliar symptom expression after feeding by leafhoppers transferred from ALuY symp- tomatic lucerne plants. Twelve of 25 plants developed phytoplasma disease-like symptoms including stunting and yellowing. The most pronounced foliar symptoms were displayed by five plants that had been fed on by O. argentatus and four plants that had been fed on by A. torrida. One plant, fed on by O. argentatus, showed the distinctive root symptoms of ALuY. A phytoplasma was identified by electron microscopy in two plants fed on by O. argentatus and one by A. torrida. For each group of plants that had been fed on by a single leafhopper species, one plant was phytoplasma positive as determined by the polymerase chain reaction (PCR) using universal primers. The phytoplasma detected by PCR in the plant fed on by A. torrida was identified by restriction fragment length polymorphism (RFLP) analysis as the tomato big bud (TBB) phytoplasma. The PCR product from two plants fed on by B. angustatus and O. argentatus were too faint for RFLP analysis. PCR assays were conducted on DNA extracted from the head and thorax of each leafhopper species from transmission tests and from field-collected insects, but no phytoplasma DNA was detected. These findings suggest O. argentatus is a vector of the ALuY pathogen and A. torrida is a vector of the TBB phytoplasma. Key words Austroagallia torrida, Batracomorphus angustatus, Orosius argentatus, PCR, RFLP, transmission. INTRODUCTION Phytoplasmas are associated with a large number of plant diseases around the world (Marcone et al. 1997; Davis & Sinclair 1998; Khadhair et al. 1999; Schneider et al. 1999). Symptoms broadly associated with phytoplasmas include witches’ broom, little leaf, stunting, phyllody and big bud (McCoy 1979). Insects that have been shown to be responsible for the vectoring of phytoplasmas are leafhoppers (Membra- coidea), planthoppers (Fulgoroidea) and psyllids (Psylloidea) (Ploaie 1981; Hill & Sinclair 2000). Australian lucerne yellows (ALuY) is a serious disease causing an estimated annual loss of $7 m to the lucerne seed industry (Pilkington et al. 1999) and has been reported in Australian lucerne stands since the early 1950s (Anonymous 1953). In 1979, Australian lucerne yellows (ALuY) levels were very low amongst crops re-sown after damage from exotic aphids (McGechan 1980). Levels of damage increased in 1980 (McGechan 1980) and since then has increased in intensity to current levels (Pilkington et al. 1999). The plan- thopper family Delphacidae and the cicadellid subfamily Deltocephalinae contain the greatest number of documented phytoplasma vector species (Fletcher 1984). Fletcher (1980) conducted insect-trapping studies in order to identify the vec- tors responsible for the spread of ALuY, and analysis of the occurrences of the 26 cicadellid species collected in that study indicated that 23 were not associated with the disease. The three possible vector species for the disease were to undergo subsequent transmission tests, though these tests were not undertaken and we could find no evidence of other similar studies in the current literature. Symptoms of ALuY include yellow discolouration of foli- age that is typically consistent over the entire plant (Stovold 1983; Pilkington et al. 1999, 2002). The plant also expresses a distinctive yellow to dark brown discolouration immediately under the periderm of the tap-root (Stovold 1983; Pilkington et al. 2002). ALuY disease is associated with a phytoplasma (Pilkington et al. 2002, 2003a). Three leafhoppers, Orosius argentatus (Evans), Austroagallia torrida (Evans) and and Batracomor- phus angustatus (Osborn), have a spatio-temporal correlation with disease symptoms and are possible vectors of the ALuY pathogen (Pilkington et al. 2003b). To date, however, there has been no direct experimental evidence to implicate any of the species as ALuY vectors.