RAPID COMMUNICATION Phytopathogen Lures Its Insect Vector by Altering Host Plant Odor Christoph J. Mayer & Andreas Vilcinskas & J��rgen Gross Received: 11 May 2008 /Revised: 2 June 2008 /Accepted: 5 June 2008 /Published online: 4 July 2008 # Springer Science + Business Media, LLC 2008 Abstract Many phytopathogens that cause worldwide losses of agricultural yield are vectored by herbivorous insects. Limited information is available about the inter- actions among phytopathogens, host plants, and insect vectors. In this paper, we report that the cell wall-lacking bacterium Candidatus Phytoplasma mali can alter both the odor of its host plant (apple) and behavior of its vector, the univoltine psyllid Cacopsylla picta. Apple trees infected by this phytoplasma emitted higher amounts of ��-caryophyl- lene when compared to uninfected ones. Psyllids that had no previous contact with Ca. P. mali, as well as infected pyllids, are more attracted by volatiles emitted from phytoplasma-infected apple plants than from uninfected ones. Psyllids that had developed on infected plants without getting infected showed the opposite behavior. These results suggest that the pathogen modifies host plant odor that lures its vector to infected plants. This may result in higher numbers of transmitting vector insects within the population. Keywords Apple proliferation . Vector���plant���pathogen interaction . ��-Caryophyllene . Candidatus Phytoplasma mali . Cacopsylla picta . Malus domestica Introduction The cell wall-lacking bacterial phytopathogen Candidatus Phytoplasma mali causes the apple proliferation disease (Seem��ller and Schneider 2004) and is responsible for severe economic losses by inducing ���witches brooms��� and tasteless dwarf fruits in apple trees (Malus domestica), for which no curative approaches are available (Seem��ller et al. 2002). The univoltine psyllid species Cacopsylla picta (Foerster) (Hemiptera: Psyllidae) was discovered as a vector of this phytoplasma that infects both the host plant and insect vector (Frisinghelli et al. 2000). Information on orientation and host finding by these phloem-feeding insects vectoring phytoplasmas is lacking (Weintraub and Beanland 2006). The olfactory response of two psyllid species suggested that plant kairomones may play a role in host-finding behavior (Gross and Mekonen 2005), but the influence of the vectored phytoplasma in this multitrophic system has not been examined in detail. In this study, we investigated the behavior of both infected and uninfected vector insects toward the odors of uninfected and phytoplasma infected plants in a Y- shaped olfactometer. The infection status of psyllids and plants was confirmed by polymerase chain reaction (PCR) with primers specific to Ca. P. mali. Further, we collected the headspace of uninfected and infected apple plants for a period of 45 d and analyzed the volatile components by gas chromatography/mass spectrometry (GC/MS). Materials and Methods Cultivation of Plants Two-year-old apple trees (cultivar Gala Royal on rootstock M9) were either infected with J Chem Ecol (2008) 34:1045���1049 DOI 10.1007/s10886-008-9516-1 C. J. Mayer : A. Vilcinskas : J. Gross Institute for Phytopathology and Applied Zoology, Justus-Liebig University of Giessen, Gie��en 35392, Germany C. J. Mayer : J. Gross (*) Julius Kuehn Institute, Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Fruit Crops and Viticulture, Dossenheim 69221, Germany e-mail: juergen.gross@agrar.uni-giessen.de

Ca. P. mali by grafting shoots of already infected apple trees (from the previous years experiments) or left untreated as uninfected controls. All trees were checked for apple proliferation prior to experiments by extracting deoxyribonucleic acid (DNA) of the rootstock���s phloem followed by PCR with specific primers (fAT/rAS) as described below. Trees were transferred from a cooling chamber to a greenhouse chamber 5 wk before the headspace sampling was started. The trees were maintained under natural light and temperature conditions (day/night: 16/8 hr, 20/15��C, 60% relative humidity [RH]) until flowering was completed (phenology stage 68). Subse- quently, trees were transferred into a climate chamber (day/ night: 12/12 hr, 20��C constant, 60% RH) for headspace sampling (phenology stages 69���79). Rearing of Insects for Bioassays Mature adults of C. picta were collected from apple trees (M. domestica) in early spring after having returned from their overwintering host plants. In rearing cages (60��60��90 cm), they were maintained on uninfected or Ca. P. mali-infected apple plants where they could feed and oviposit. The rearing cages were located in a tempered greenhouse chamber under natural light conditions and a day/night temperature program (20/15��C). For behavioral trials, test insects were collected from the boxes 1 d before testing in groups of ten in Eppendorf vials at 4��C. All insects were tested within 2 wk after emergence. Behavioral Bioassays Behavioral tests were carried out by using a dynamic olfactometer consisting of a Y-shaped glass tube (entrance arm: 12.5 cm, test arms=21.0 cm, inner diameter=0.6 cm) mounted on an angular board. Charcoal cleaned air (granulated 4���8 mm, Applichem GmbH, Darmstadt, Germany) was pumped (75 ml/min) through two glass jars (vol=2 l) containing the volatile sources. A twig of uninfected or phytoplasma infected apple plants (length 10���15 cm, phenology stage 69���71) was placed in a water-filled glass vial in each jar. The odors of infected and uninfected twigs were offered simulta- neously. According to the method of Soroker et al. (2004), for each test, ten females were put together into the entrance arm. Every individual that passed a final marking (10.0 cm after the branching) on one of the test arms within 15 min was counted and placed into separate vials filled with ethanol (70%) for later determination of their infection status (see below). Both experiments were repeated 29 times. The numbers of psyllids were analyzed statistically after log (x+0.5) transformation by dependent paired t test using Statistica 5.5. DNA Extraction: Plant Material Phloem tissue from cut roots was abraded. Between 1.0 and 1.5 g of phloem tissue was subjected to DNA extraction following the procedure described by Doyle and Doyle (1990). Instead of 0.2% ��- mercaptoethanol, 2% sodium metabisulfide was used. The resulting DNA pellet was resuspended in 50 ��l of sterile water and stored at -20��C. Insect Material Psyllids that were used in bioassays (see above) were subjected to DNA extraction by using the same protocol as described above for plant material. Single insects were homogenized with a conical pestle (polypropylene, 1.5 ml, Eppendorf, Hamburg, Germany) in a 1.5-ml tube containing 150 ��l extraction buffer and a pinch of silicon carbide (carborundum, Sigma-Aldrich, Munich, Germany) as grinding additive. The resulting DNA pellet was resuspended in 20 ��l sterile water and stored at -20��C. PCR Analysis DNA was purified by using the primer pair fAT/rAS specific for Ca. P. mali (Smart et al. 1996) and amplifying a 400-bp sequence in the 16S���23S ribosomal ribonucleic acid spacer region. PCR reactions were per- formed in a thermal cycler (Robocycler 96, Stratagene, La Jolla, CA, USA) with a reaction volume of 25 ��l containing 2.5 ��l extracted DNA, 0.5 pM of each primer, 250 ��M of each nucleotide, 0.5 U polymerase, and 1�� polymerase buffer (Invitrogen, Karlsruhe, Germany). Samples were subjected to 35 cycles that consisted of 1 min denaturation at 95��C, 1 min annealing at 51��C, and 1.5 min at 70��C. The products of each PCR were electrophoresed on a 1% agarose gel containing ethidium bromide (0.3 ��g/ml) in 1�� TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8.0). DNA was visualized and photographed while exposed to UV light (302 nm). Headspace Sampling Single branches of apple plants (see above) were carefully wrapped in polyethylene terephthal- ate bags (20 cm diameter, Melitta, Minden, Germany). A stream of purified air (250 ml/min controlled by a flowmeter) was pumped through the bag for 4 hr. For each treatment, two trees were sampled every second or third day over a period of 45 d. Volatiles from plant headspace were trapped in collection filters (charcoal 5 mg, Gr��nicher+Quartero, Daumazan, France) and eluted by rinsing the filter with 25 ��l of dichloromethane containing 50 ng/��l of tridecane (Sigma-Aldrich, Munich, Germany) as internal standard (IS). Analysis of Headspace Samples by GC/MS Each sample (0.5 ��l) was injected splitless into a gas chromatograph (Shimadzu GC 17A, injector temperature 300��C) equipped with a 30 m��0.32 mm��0.25 ��m HP-5 column (J & W, Santa Clara, CA, USA). The temperature program started at 40��C, was held for 3 min, and then 1046 J Chem Ecol (2008) 34:1045���1049