Effects of phosphorus deficiency and genetic variation on leaf terpene contents and 2 emission rates in Pinus pinaster seedlings susceptible and resistant to the attack of the 3 pine weevil

33 We studied the effects of phosphorus fertilization on foliar terpene concentrations and 34 on foliar volatile terpene emission rates in different half-sib families of Pinus pinaster 35 Ait. seedlings. Half of them appeared to be resistant to the attack of the pine weevil 36 Hylobius abietis L., a generalist phloem feeder, and the other half appeared to be 37 susceptible to this insect. We hypothesized that P stress could modify the terpene 38 concentration in the needles and thus derive to altered terpene emission patterns 39 relevant to plant-insect signalling. The total concentrations and emission rates ranged 40 between 5732 and 13995 μg g d.m. and between 2 and 22 μg g d.m. h 41 respectively. The storage and emission were dominated by the isomers α and β42 pinene (77.2 % and 84.2 % of the total terpene amount respectively). P stress caused 43 in both resistant and susceptible families an increase of 31% of the foliar terpene 44 concentrations with an associated 5-fold decrease of the terpene emission rates. Those 45 higher contents would indicate an allocation of the “excess of carbon” generated due 46 to growth being limited because of P scarcity, to terpene emissions. Sensible families 47 showed a higher increase of terpene emission rates, which could be related to plant48 animal communication. 49 50 Introduction 51 Phosphorus has many roles in plant growth and metabolism. One of the principal 52 functions of phosphorus is energy transfer: through the action of adenosine 53 triphosphate (ATP). ATP and its derivatives, ADP and AMP, are involved in all 54 aspects of energy transfer in every part of plant growth. Apart from this global 55 function, phosphorus is also necessary for assembling nucleic acids (DNA and RNA), 56 proteins, enzymes and carbohydrates. It plays an essential role in photosynthesis and 57 is involved in the formation of sugars and starch. The various roles of phosphorus 58 denote the fact that it is important in the formation of seeds and the development of 59 roots. It also speeds plant maturity and helps the plant resist stresses (Urbano, 1999). 60 However, fertilization of young pine seedlings and the subsequent boosting of 61 primary growth rates could lead to increased susceptibility to pests and diseases due 62 to altered allocation patterns of energy to growth and defence and/or improved tissue 63 quality for the insects. In this sense, in a field study Zas et al. (2006a; 2008) found 64 that traditional silvicultural practices such as phosphorus fertilization could lead to 65 greater susceptibility to the attack by the pine weevil Hylobius abietis L. in seedlings 66 of P. pinaster and P. radiata, which may be at least partially explained by a reduction 67 in resistance (Moreira et al. 2008). The pine weevil Hylobius abietis L. is a generalist 68 phloem-feeder that constitute a major pest in conifer plantations in all Europe, where 69 causes important regeneration problems due to the fact that adults feed the bark of 70 young seedlings (Conord et al. 2006; Leather et al. 1999). The susceptibility of P. 71 pinaster to H. abietis attack has been found to present an intense genetic variation, 72 where some families were consistently more damaged than others (Zas et al. 2005). 73 Greater nutrient availability could directly increase the nutritional value of the 74 plant tissues and thus increase the preference by the insects (Ayres & Lombardero 75 2000; Moreira et al. 2009). Phosphorus fertilization on P stressed pine seedlings may 76 diminish the allocation of energy to constitutive and induced defences by favouring 77 the growth rates. Several models of plant defence suggest altered patterns of 78 allocation to chemical defences in environments with increased nutrient availability. 79 The Carbon nutrient balance (Bryant et al. 1983) stated that when growth is limited 80 by nutrients, plants allocate the “excess carbon” to the production of secondary 81 metabolites. The Growth differentiation balance (Lorio 1986) recognizes that all 82 secondary metabolites have an ontogenetically determined phenology and that their 83 synthesis is emphasized during periods of plant differentiation. Growth dominates 84 during favourable conditions, and differentiation is at a maximum only when 85 conditions are suboptimal for growth. This could be more evident in tree species with 86 predeterminated growth such as pine trees. The Optimal allocation model (Tuomi et 87 al. 1991) predicts decreasing investment in defence with increasing resource 88 availability, because reduced costs of tissue production could compensate higher risks 89 of herbivore predation. Higher phosphorus availability could also lead to a higher 90 appearance of the fertilized plants to the insect. Fertilization could change the amount 91 of emitted and leaf-contained volatile organic compounds as it may affect the 92 secondary metabolism as stated by “excess carbon” hypotheses (Peñuelas & Estiarte 93 1998). 94 Maritime pine (Pinus pinaster Ait.) has been widely chosen as forestation 95 species in Galicia (NW Spain) since the XVIII th century. Despite being partly 96 replaced in the last decades by species with higher productions like Pinus radiata and 97 Eucalyptus globulus, P. pinaster is still the most important forest tree species in 98 Galicia. According to the last forest survey (DGCN 2000), Galicia has 389,489 ha of 99 monospecific stands and 243,735 ha of mixed stands with eucalyptus or broad-leaved 100 species. Thus, P. pinaster is present in 44% of the total Galician wooded area. The 101 intensive silviculture applied to P. pinaster entails short rotations (15 to 45 years), in 102 which there is an important extraction of nutrients of the system (Merino et al. 2003). 103 Conifer plantations in Galicia commonly suffer important nutrient deficiencies 104 (Martins et al. 2009). These plantations are usually located on acid soils with few 105 nutrients, especially phosphorus. Moreover, the loss of nutrients due to harvesting can 106 lead to decreased reserves of soil available limiting nutrients (Dambrine et al. 2000; 107 Merino et al. 2005). Under those conditions, phosphorus stress is commonly found in 108 conifer and especifically in P. pinaster stands in NW Spain (Martins et al. 2009). 109 We hypothesized that P stress could modify the terpene concentration in the 110 needles and the photosynthetic activity of P. pinaster thus leading to altered terpene 111 emission patterns relevant to plant-insect signalling. The objective of the present 112 study was to test this hypothesis. With this aim and the additional aims of studying the 113 effect of genetic variation and the relationships with the resistance to pests, we 114 analyzed the effect of P fertilization on terpene concentrations and on terpene 115 emission rates in half-sib families of P. pinaster seedlings cultivated under controlled 116 conditions, previously found to be resistant or susceptible to the large pine weevil in 117 field conditions in Galicia forests. 118