1. Effects of microbial symbionts on aboveground plant-insect interactions in Plantago lanceolata Royal Netherlands Academy of Arts and Sciences Arjen Biere Netherlands Institute of Ecology (NIOO-KNAW) Wageningen

2. Microbial mediation of Plant-Insect interactions Microbe Barbosa, Krischick & Jones (1991) Biere & Bennett (2013) Funct Ecol Endosymbionts Resource exploitation Insect Plant Mycorrhiza PGPR / PGPF Rhizobia Endophytes Resource quality Arbuscules Mycorrhizae trade N/P for C

3. Pozo & Azcon-Aguilar (2007) COPB Jung & Pozo (2012) J Chem Ecol Parasitoids Priming of JA- signaled defense Repression of SA- signaled defense Mycorrhizae affect defense against aboveground insects Necrotrophic fungi Generalist chewing insects Bacterial pathogens Galling insects - Mycorrhiza- Induced Resistance Soil-borne pathogens Root-feeding insects Parasitic nematodes - Mycorrhiza- Induced Susceptibility Biotrophic fungi, viruses Specialist chewing insects Phloem-feeding insects +

4. Do mycorrhizal effects on herbivore growth vary with plant genotype & (nutrient, P) environment ? Insect Mycorrhiza Mycorrhizal effects on aboveground herbivores Plant size, Leaf C/N Induction/priming of plant secondary metabolites Plant VOCs Spodoptera exigua Funneliformis mosseae Plantago lanceolata How do mycorrhizae alter leaf C/N & secondary metabolites ? How does that affect herbivore growth ? O HOH 2 C Gluc OH aucubin O HOH 2 C Gluc OH catalpol Iridoid glycosides (IG) Plantago lanceolata Do effects of mycorrhiza interact with those of previous herbivory ?

5. Plant Genotype1 Plant Genotype2 Low P High P (0.3%) (0.03%) T=48 Harvest Bioassay, VOC 2 Phosphate x 2 Genotype x 2 Mycorrhiza x 2 Herbivory x 10 Replicates = 160 plants Control T=0: Mycorrhiza T=45: Herbivory (24h) Herbivory Myc+Herb Experimental set-up

6. Contr /Geno1 Contr /Geno2 0 2 4 6 8 10 Leaf IG (%) Low P High P G*** 0.00 0.05 0.10 0.15 0.20 0.25 0.30 larval ECI Low P High P G* P*** GxP* 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 larval RCR Low P High P G** P** 0 5 10 15 20 25 30 35 40 Leaf C/N Low P High P G** P*** GxP* M*** MxG* MxGxP* MxG* 0.0 0.2 0.4 0.6 0.8 1.0 larval RGR Low P High P Mycorrhizal effects on leaf quality and insect growth Mycorrhizal effects on leaf consumption (RCR) differ between plant genotypes (they swamp genotypic differences in resistance !) Despite their positive effects on leaf quality (decreasing C/N, IG), mycorrhizae do not enhance insect RGR (induction of unmeasured defense compounds ?) High P: Mycorrhizae decrease leaf IG increase leaf N >> higher RGR ? MxP* Myco / Geno1 Myco / Geno2

7. (Previous) Herbivory Control Mycorrhiza 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 RCR High P v Herb+Myc Genotype1 Interactive effects of mycorrhizae and (previous) herbivory Mycorrhiza & herbivores suppress each other’s negative effects on RCR of later arriving herbivores. MxH*

8. Mycorrhizal effects on VOC & parasitoid attraction Biere & Boom, unpubl. 0 100 200 (Z)-3-hexenyl acetate GLV (Rhizophagus /JA) 0204020406080 Biere & Boom, unpubl. Funneliformis /Chrysodeixis/ Microplitis Myc Herb Herb+ * (Z)-3-hexenyl acetate GLV (E)-β- caryophyllene E-β- farnesene SqTRP Volatile emission (ng/g dw/ hr) 0 100 200 300 400 500 600 700 Fontana et al. (2009) J Chem Ecol* (Rhizophagus /Spodoptera) Control Mycorrhiza Herbivory Myc + Herb * See also Babikova et al (2014) Funct Ecol; but see Schausberger et al (2012) Funct Ecol Parasitoid prefers (damaged) non-mycorrhizal plants over (damaged) mycorrhizal plants Speculation: low parasitoid preference for mycorrhizal plants due to suppression GLV / sqTRP Y-tube olfactometer

9. Conclusions Mycorrhizae (like other plant-associated microbes) can be important “hidden players” in plant-insect interactions. Despite their positive effects on leaf quality (lower C/N, IG), mycorrhizae do not necessarily enhance insect larval growth. In one case, mycorrhizae suppress (not enhance) the plant’s ability to induce defense in response to herbivory. In one case, mycorrhizae reduced (not enhanced) attractiveness of damaged plants to parasitoids. Due to suppression of GL/terpenoid HIPVs ?

10. Acknowledgements Thanks to: -Timo Boom, Dylan Kolenberg, Ciska Raaijmakers, Kees Hordijk, Iris Chardon, Rita Gols -Terrestrial Ecology colleagues at NIOO

12. 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 1020304050 0 44*** 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 014816 2 0.0 2.0 4.0 8.0 014816 2 6.0 Leaf % IG 3* 11** 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 00.512648 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 00.512648 0 12648 0.0 2.0 4.0 8.0 6.0 00.512648 0.0 2.0 4.0 8.0 6.0 Leaf % aucubinLeaf % catalpol 8*** 0 3*17*** 0.0 2.0 4.0 8.0 6.0 1020304050 0 19*** Leaf C/N Larval ECI Larval RCR 0.0 0.2 0.4 0.6 0.8 1.0 1020304050 0 Larval RGR 9*** Leaf C/N 014816 2 0.0 0.2 0.4 0.6 0.8 1.0 15*** Leaf % IG 00.512648 0.0 0.2 0.4 0.6 0.8 1.0 00.512648 0.0 0.2 0.4 0.6 0.8 1.0 14***9*** Leaf % aucubinLeaf % catalpol General patterns in effects of leaf traits on insect growth Low leaf quality (high C/N) is only partly compensated by increased feeding rate IGs mainly function as feeding deterrents, but aucubin also reduces digestibility

13. Plant dry wt (g) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Low P High P G* P* GxP* Low IG High IG Mycorrhiza plants: M*** MxP*** Plant genotype, soil P, and mycorrhizal effects on insect growth

14. AMF suppresses IG induction by the herbivore AMF suppression Leaf iridoid glycosides (%dw) 0.5 0.4 0.3 0.2 0.1 CMHMHMH Glomus “white”’ Effects of AMF on aboveground IG levels Bennett, Bever & Bowers (2009) Oecologia MHMH Herb + Myc C Contr H Herb - Consistent across plant genotypes ? - Effect on (later arriving) herbivore ? M Myc Scutellospora callospora