Indirect Two-Way Interactions between Aphids and a Pathogen on

Indirect Two-Way Interactions between Aphids and a Pathogen on Roses N. Desneux, R. Mouttet, P. Bearez and C. Poncet INRA Unité de Recherches Intégrées en Horticulture UR880, 400 Route des Chappes 06903 Sophia-Antipolis France Keywords: indirect interactions, induced plant responses, Rhodobium porosum, Botrytis cinerea, integrated pest management Abstract Indirect interactions between plant pathogens and phytophagous pests can occur when infestation by an attacker alters the shared host in a way that affects another attacker that could be spatially or temporally separated from the first one. The most commonly invoked scenario involves the induction of plant defence reactions which primarily depend on characteristics of both pathogens and herbivores considered. We carried out experiments under controlled conditions on young rose plants in order to assess the impact of such indirect interactions on life history traits of two pests on rose: a necrotrophic fungus, Botrytis cinerea, and an aphid, Rhodobium porosum. We highlighted (1) an indirect two-way negative interaction between B. cinerea and R. porosum which is expressed by a reduction of aphid growth rate and a decrease in growth speed of fungal lesions on the plants, and (2) a negative indirect effect of B. cinerea on insect’s behaviour, as aphid preferred B. cinerea-free plants than B. cinerea-infested plants when given a choice. The implications of results in Integrated Pest Management on rose and mechanisms involved in the interactions are both discussed. INTRODUCTION Species of an agroecosystem may interact with one another, either directly or indirectly, in numerous and complex ways (Wootton, 1994). Indirect interactions have been somewhat neglected despite their diverse (positive, negative or neutral) effects on populations (Barbosa et al., 1991). For instance, indirect interactions between plant pathogens and phytophagous insects can occur when infestation by a first attacker induces changes to the shared host that affect a second attacker which is spatially and/or temporally separated from the first one (Hatcher, 1995; Rostas et al., 2003). If one considers the presence of multiple pests on a plant as a standard in agroecosystems, such interactions are likely to occur (Fournier et al., 2006). The induction of plant defence reactions is thought to be deeply involved via which signalling pathways and secondary metabolites production have predominant roles (Paul et al., 2000; Biere et al., 2004). Two major signalling pathways are involved: infestation by biotrophic pathogens or attack by sucking insects typically activates the salicylic acid (SA) pathway and results in systemic acquired resistance (SAR) against plant diseases, while attack by herbivores or necrotrophic pathogens usually triggers the jasmonic acid (JA) pathway (Pieterse and Dicke, 2007; Smith et al., 2009). Crosstalks between signalling pathways and fine-tuning defence responses complicate this basic framework and might shape the outcome of interactions between pathogens and insect herbivores (Hatcher et al., 2004; Taylor et al., 2004). In such a context, our project focused on the assessment of indirect interactions between two rose pests: a necrotrophic pathogen, Botrytis cinerea (Pers.: Fr.), and a piercing-sucking insect, Rhodobium porosum (Sanderson). Main objectives consisted in the definition of interaction patterns and evaluation of their effects on the population dynamics of attackers. Working hypotheses were as follow: (1) indirect interactions are likely to occur between attackers inducing similar plant defence reactions; (2) the spatiotemporal design of attacks may impact the outcome of interactions. From this Proc. XXVIIIth IHC – IS on Greenhouse 2010 and Soilless Cultivation Ed.: N. Castilla Acta Hort. 927, ISHS 2012

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perspective, we realized cross infestation experiments associated to behaviour experiments in order to test the impact of an infestation by a first attacker on the performance and behaviour of a second attacker, with special attention being paid to the spatiotemporal distribution of infestations. Spatial aspects (distance between attack events) may be of importance as the plant is expected to show stronger responses locally than systemically (Durrant and Wong, 2004) while temporal aspects (delay between attacks) might determine the resistance state of the plant (Scheideler et al., 2002) Experiments were undergone on a woody plant model, Rosa hybrida L. Therefore our study is a first step in understanding the effects of indirect interactions on a plant type whose defence strategy differs greatly from herbaceous model plants (Herms and Mattson, 1992). MATERIALS AND METHODS Rearing of rose plants, Botrytis and aphids The plants used in the experiments were young rose plants, Rosa hybrida ‘Sonia’; a cultivar which is known to be vulnerable to Botrytis cinerea (J.P. Onesto, pers. commun.). They were grown in climatic chambers (24±1°C, RH: 65%, photoperiod 16L:8D) with a daily application of nutrient solution. Prophylactic measures were applied to prevent the presence of any pest in the climatic chambers, so that plants used for the experiments had never been into contact with plant pathogens or herbivores. R. porosum individuals were reared on caged rose plants (R. hybrida ‘Sonia’) in climatic chambers (24±1°C, RH: 65%, photoperiod 16L:8D). Spores of B. cinerea were obtained from mycelium cultivation of isolate T4 (J.P. Onesto, pers. commun.) on maltagar Petri dishes, after the growing mycelium had covered the entire Petri dish surface. Spore suspensions were prepared by homogenizing the Petri dish content with a Potato Dextrose Broth solution. Preliminary results showed that using Potato Dextrose Broth Solution was much more efficient than using dry spores. After centrifugation and resuspension, spore concentration was measured in a Mallasez® cell and adjusted to a concentration of 105 spores per ml. Cross Infestation and Behaviour Experiments Cross infestation experiments consisted in a plant infestation by a first attacker, pathogen or insect, followed by a second infestation by another attacker, insect or pathogen respectively. Attacks were separated in time and space. Regarding the spatial design, infestations were either local or systemic. Local ones were realized on two leaflets from the same leaf whereas systemic ones were realized on two leaflets from different leaf stages. Clip cages were used to constrain the attackers on their respective leaflet (Desneux and O’Neil, 2008; Ramirez-Romero et al., 2008). They were composed of a Petri dish cover (D: 43 mm) whose extremity was covered with foam ring, the whole being attached to a fiberglass stake. Insect clip cages presented a circular opening made of insect-proof mesh. Regarding the temporal design, the dissociation between attacks was either of short term (1 day) or of long term (7 days). Infestations by the pathogen were realized by dropping off 10 µl of spore suspension on the upper leaf surface. For first attackers, pre-infestations by insects were realized by placing ten aphids on the lower leaf surface. For second attackers, infestations by insects consisted in placing three adult aphids on the lower leaf surface. A performance parameter of the second attacker was then assessed and compared to the control situation with no pre-infestation. The lesion size of the pathogen and the number of aphids were the two parameters of interest. They were evaluated four days following the second infestation. For the experiments, plants were selected for equal size, arranged in a randomized way. Behaviour experiments consisted in dual choice experiments. Ten aphids were offered two leaf discs (D: 25 mm) within Petri dishes (D: 53 mm) and allowed to settle on for 15 min. Leaf discs were punched out from healthy leaves or leaves pre-infested for four days by B. cinerea either locally or systemically. The number of insects present on each disc after 15 minutes was then recorded. 238

Statistical Analysis Normality of data was first assessed by a Shapiro-Wilk test. For cross infestation experiments, pathogen lesion sizes and numbers of aphids were compared within the different spatial distributions (spatial factor) using generalized linear models (GLM) with a Poisson distribution and a log link function. They were followed Tukey’s post hoc tests for multiple comparisons inside temporal sub datasets. Data from dual-choice assays were examined with Wilcoxon rank-sum tests. Statistical analyses were performed under the R software. RESULTS Pre-Infestation with Botrytis The growth of aphid population was significantly affected by pre-infestation of host plants by the pathogen B. cinerea (P