Artificial infection of Vaccinium vitis-idaea L. and defence responses to Exobasidium species Tuija Pehkonen a , Janne Koskima ¨ki a , Kaisu Riihinen b , Anna Maria Pirttila ¨ a , Anja Hohtola a , Laura Jaakola a , Anne Tolvanen c, * a Department of Biology, University of Oulu, FIN-90014, Oulu, Finland b Department of Biosciences, University of Kuopio, P.O.B.1627, FIN-70211 Kuopio, Finland c Finnish Forest Research Institute, Muhos Research Unit, Kirkkosaarentie 7, 91500 Muhos, Finland article info Article history: Accepted 15 August 2008 Keywords: Boreal Exobasidium splendidum Exobasidium vaccinii Flavonoids Inoculation Lingonberry Subarctic Vaccinium vitis-idaea abstract An inoculation method for Exobasidium splendidum and Exobasidium vaccinii was developed on the dwarf shrub Vaccinium vitis-idaea. Using inoculated ramets, we investigated whether there are differences between V. vitis-idaea populations in the susceptibility to Exobasidium infections and whether the defence reaction of V. vitis-idaea is visible at a molecular level. Sixteen V. vitis-idaea clones from four populations were propagated in tissue cultures and the ramets were inoculated with E. splendidum or E. vaccinii fungi. The expression of three flavonoid biosynthetic genes (chalcone synthase, dihydroflavonol 4-reductase and anthocyanidin synthase) and the accumulation of flavonoids and hydroxycinnamic acids were determined in response to E. splendidum infection. A pathogenesis-related (PR 4) gene was isolated and its expression was studied in host ramet leaves. To our knowledge, this was the first successful artificial infection reported with E. splendidum. Disease frequencies of the inoculated ramets were between 32% and 47% for E. splendidum and 33% for E. vaccinii, but below 10% in uninoculated control ramets. There were no differences in disease frequencies between V. vitis-idaea populations. Both symptomatic leaves and healthy leaves of diseased ramets showed activation of flavonoid biosynthesis at the gene level, whereas expression of PR 4 was observed only in symptomatic leaves. The increase of flavonoid biosynthesis in healthy leaves of diseased ramets may represent a general response to stress or a role in defence against the pathogen E. splendidium. Ability of V. vitis-idaea to defend chemically against Exobasidium fungi and the heterogeneity of genotypes, age, size, and growth rates in host plant pop- ulations might be reasons for the low infection incidence of Exobasidia in nature. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Both environmental and genetic factors are known to influence the susceptibility of plants to pathogen infections [1–3]. When the pathogen infections are scattered, however, it is difficult to inves- tigate which factors have an impact on the disease frequencies on plants in nature. Experimental work and pathogen inoculations are therefore needed. In this study, a dominant boreal-subarctic dwarf shrub Vaccinium vitis-idaea L. and its two highly host and tissue specific biotrophic pathogens, Exobasidium splendidum Nannf. and Exobasidium vaccinii Woron (Basidiomycotina) were investigated. E. splendidum is a monocyclic fungus, i.e. it reproduces only once during the growing season [2]. Dormant spores overwinter on vegetative buds and infect the newly developing shoots in spring. Infections by E. vaccinii are found from early until late season, which indicate that the species reproduces several times during the growing season. E. splendidum is restricted to high latitudes and altitudes, whereas E. vaccinii follows its host in most of its area except the highest latitudes and altitudes [2]. Environmental factors are essential in determining the frequency of Exobasidium infections in V. vitis-idaea [1,4,5], whereas there is no information on genetic differences between V. vitis-idaea populations with respect to their susceptibility to Exobasidium infections. Since the natural infection rate is less than 5% for E. splendidum and less than 10% for E. vaccinii [4], other factors than environment probably also limit their distribution. One of these factors may be the ability of V. vitis-idaea to defend chemically against the pathogen. Exobasidium species infect immature, vegetative and reproduc- tive plant tissues [1,2], which die after the sporulation at the end of the growing season. Symptoms of E. splendidum are slightly elon- gated shoots and moderately enlarged, bright red leaves. E. vaccinii * Corresponding author. Tel.: þ358 10 211 3782; fax: þ358 10 211 3701. E-mail addresses: tuija.pehkonen@inari.fi (T. Pehkonen), janne.koskimaki@ oulu.fi (J. Koskima ¨ki), kaisu.riihinen@uku.fi (K. Riihinen), am.pirttila@oulu.fi (A.M. Pirttila ¨), anja.hohtola@oulu.fi (A. Hohtola), laura.jaakola@oulu.fi (L. Jaakola), anne.tolvanen@metla.fi (A. Tolvanen). Contents lists available at ScienceDirect Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp 0885-5765/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2008.08.002 Physiological and Molecular Plant Pathology 72 (2008) 146–150