The preference of the C. populi beetles for non-inoculated poplars is suggested to be caused by EMF induced molecular changes in the leaves. Mycorrhizal associations of plants can influence metabolites in the leaves. For example, the production of iridoid glycosides was induced in leaves of AMF-inoculated Buckhorn plantain plants (Plantago
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lanceolata L.) (Fontana et al., 2009). Gange and West (1994) observed an increased level of the feeding deterrents aucubin and catalpol in the leaves of AMF-inoculated compared to non-inoculated P. lanceolota and a reduced growth rate of the Garden tiger moth (Arctia caja) on mycorrhizal plants.
The inoculation of poplar with L. bicolor affected the leaf tanscriptome, but of only few genes. Recently, the response of P. × canescens to the EMF P. involutus was studied at the transcript level in EM roots and the transcriptional response of only 0.3% genes was changed (Luo et al., 2009a). This intensity found in the roots agrees with the response to the EM inoculation in poplar leaves, observed in the present study. Here, the EMF inoculation affected the transcript abundance in distinct pathways in the leaves such as hormone signalling pathways, flavonoid biosynthesis pathways and aldoxime biosynthesis.
Inoculation of poplars with L. bicolor decreased the transcript abundance of genes in the ABA signalling pathway. Hormonal changes in mycorrhizal plants were expected and have been detected in below and aboveground parts of mycorrhizal plants with contrasting reports about ABA. An increased transcript abundance of genes related to ABA was detected in roots and a decreased abundance in shoots of AMF-inoculated tomato plants (Solanum lycopersicum L.) (Fiorilli et al., 2009). AMF-inoculated S. lycopersicum plants had lower ABA levels in the leaves (Fiorilli et al., 2011) and EMF-inoculated poplars increased ABA levels in the roots (Luo et al. 2009a). An involvement of ABA in the development of arbuscules was shown using ABA-deficient tomato mutants (Lycopersicon esculentum) that were less susceptible to AMF (Herrera-Medina et al., 2007). ABA also plays important roles in plant development, signalling and response to biotic and abiotic stress (Lee and Luan, 2012).
In the present study, the transcriptional results indicated a decreased abundance of genes in the flavonoid biosynthesis pathway, which was supported by metabolomic analysis (Kaling, personal communication). Decreased concentrations of 35 flavonoids such as catechin, dihydrokaempferol, dihydromycetrin and quercetin were detected in inoculated compared to non-inoculated poplars (Kaling, personal communication). The decreased transcript abundance of genes in flavonoid biosynthesis pathway may be related to that of the ABA signalling pathway. In Orthosiphon stamineus Benth (Cat's Whiskers) the ABA concentration was found to be positively correlated with the biosynthesis of flavonoids by analysing the production of secondary metabolites under different levels of ABA applications (Ibrahim and Jaafar, 2013).
Effects of a symbiotic partner on flavonoids in plant roots have been demonstrated. For example, in symbioses with rhizobia, root flavonoids induce nod genes in rhizobia and are therefore important for the initiation of root nodules (Wasson et al., 2006). In AM formation, flavonoids are important signalling compounds in the AM symbiosis (Larose et al., 2002; Ponce et al., 2004). However, it was assumed that specific flavonoids are not involved in the establishment of EM associations in Scots pine (Pinus sylvestris L.) (Niemi et al., 2007). Niemi et al. (2007) investigated the content of flavonoids and polyamines in Scots pine during ectomycorrhizal formation with Suillus variegatus and found quantitative changes in flavonols, catechins and tannins but no qualitative changes of the flavonoid composition in the shoots. Flavonoids have diverse biological functions (Buer et al., 2010), which include signalling via the auxin transport (Peer and Murphy, 2007) and defence against stress, pathogens and herbivores (Treutter, 2005; Bais et al., 2006).
Flavonoids may play a role in the defence of poplars against herbivores such as C. populi.
Because the transcript abundance of genes in the flavonoid biosynthesis was decreased in inoculated poplars compared to non-inoculated plants, other defensive compounds might be responsible for deterring C. populi beetles. For example, in EMF-inoculated beech (Fagus sylvatica) mycorrhizal inoculation induced the production of defensive compounds such as tannins in roots (Beyeler and Heyser 1997).
In the present study, differences in the defence reaction in inoculated and non-inoculated poplars were related to phytoalexins in the leaves. Upon beetle attack, poplars inoculated with L. bicolor had significantly higher transcript abundances of the cytochrome P450 genes of the CYP79 family, i.e. CYP79D5, CYP79D6 and CYP79D7, compared to non-inoculated plants. These genes are involved in the synthesis of aldoximes like phenyl-acetaldoxime (Irmisch et al., 2013). As demonstrated by Irmisch et al. (2013) higher phenyl-acetaldoxime concentrations reduced the survival of gypsy moth caterpillars (Lymantria dispar), decreased the weight growth of larvae and extended the time until pupation. Therefore, an enhanced biosynthesis of aldoximes in poplar leaves may be responsible for the decreased preference of C. populi to select these leaves for consumption and as oviposition sites. To gain a deeper understanding of the connection between aldoxime emission and C. populi behaviour, aldoxime concentrations in mycorrhizal poplar leaves should be measured and the reaction of C. populi beetles to plants with modified aldoxime metabolism tested.
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Inoculation of poplars with L. bicolor also affected plant growth and biomass. Under control conditions inoculated poplars had significant lower leaf biomass and plant height increment compared to non-inoculated poplars. The finding that inoculation with mycorrhiza has a positive effect on the plant reaction to stress, but reduces growth performance agrees with the study of Luo et al. (2009b). In that study P. euphratica inoculated with the EMF P. involutus had an increased osmolality in leaves under drought conditions and an increased stress tolerance, but also a reduced plant growth compared to the non-inoculated poplars. The reasons for the reduced growth in the inoculated plants needs to be elucidated in future work.