Original article

Abietane and pimarane diterpene acid evolution in Scots needles in relation to feeding of the pine , pini L.

L Buratti JP Allais C Geri M Barbier

1 Institut de Chimie des Substances Naturelles - CNRS 91198 Gif-sur-Yvette, Cedex ; 2 Station de Zoologie Forestière - INRA Ardon 45160 Olivet, France (Received 16 February 1989; accepted 30 June 1989)

Summary - Abietane and pimarane resin acids extracted from the needles of Scots pine, Pinus sylvestris, were analysed by reverse phase HPLC followed by GC of their methyl esters, in relation to the seasons, or the age of the trees. P sylvestris is the habitual host plant of the sawfly Diprion pini (, ) and results of the analyses were correlated with the feeding pattern of this in nature. An increase in resin acid concentration was observed during the growing season, but no direct relationship could be established with the feeding preferences of these . Young contained lower levels of abietane and pimarane diterpene acids than 10 or 30 year-old pines. Previous defoliation induced an increase in the neutral fraction and, although less so, in the diterpene acids in the needles formed the following year. The observed results are discussed in relation to the development of Diprion pini larvae and to previous hypotheses from other authors concerning the antifee- dant properties of the resin acids. It is concluded that, if the abietane and pimarane diterpene acids interfere with the biology of Diprion pini, they cannot, however, be considered as the most important factors in the natural equilibria of this species. Pinus sylvestris / Diprion pini / Diprionidae / sawfly / abietane and pimarane diter- pene acids / pine foliage / seasonal average variation / antifeedant property

Résumé - Évolution des acides diterpéniques de types abiétique et pimarique dans le feuillage du pin sylvestre pinus sylvestris L ; impact de l’âge des aiguilles et des arbres, influence des défoliations passées. Conséquences pour le lophyre du pin Diprion pini L. Les acides résiniques de types abiétique et pimarique extraits des aiguilles du pin sylvestre, Pinus sylvestris, ont été analysés par HPLC sur phase inverse et CPG de leurs esters mé- thyliques, en fonction des saisons et de l’âge des arbres. P sylvestris est la plante hôte habituelle de Diprion pini (Hyménoptères, Diprionidés) et les résultats des analyses ont été corrélés à l’aptitude de cet insecte à se nourrir sur le feuillage de cet arbre. On note une augmentation du taux des acides résiniques à la belle saison, durant la phase de croissance des aiguilles, mais aucune relation directe n’a pu être observée pour expliquer les préférences alimentaires de cet insecte. Les jeunes pins contiennent un taux plus faible d’acides résini- ques de types abiétique et pimarique que les arbres âgés de 10 ou 20 ans. La défoliation

Correspondence and reprints de l’arbre induit dans les aiguilles formées l’année suivante une augmentation du taux des- lipides neutres et, à moindre titre, des acides résiniques (plus particulièrement de l’acide abiétique). Les résultats obtenus sont discutés en fonction du choix alimentaire des larves de D pini et des hypothèses émises par d’autres auteurs concernant l’action anti-appétante des acides résiniques. En conclusion, l’aptitude de D pini à consommer le feuillage du pin sylvestre, Pinus sylvestris, ne paraît pas être directement liée aux variations de sa teneur en acides résiniques de types pimarique et abiétique. Toutefois ceci n’exclut pas toute action des ces composés dans la relation D pini - Pinus sylvestris comme le suggèrent certains résultats: taux particulièrement élevé de l’acide abiétique dans le feuillage des pins un an après une défoliation et dans les aiguilles des essences non attaquées tel Pinus pinaster. Seule une étude exhaustive des composés de la fraction acide, et de leur variations nous permettra d’apprécier le rôle effectif des acides résiniques dans les interactions Diprions-Pins sylvestres. Pinus sylvestris / Pinacée / Diprion pini / Diprionidé / Tenthrède / acide pimarique / acide abiétique / aiguille de pin / feuillage / variation saisonnière / propriété anti- appétante

INTRODUCTION prion or Diprion species living on the Jack pine, Pinus banksiana, Lambert Scots pine, Pinus sylvestris L, is an (Schuh and Benjamin, 1984a, b); they economically important European pulp- were also tested on Pristiphora erich- wood and lumber conifer. Diprion pini sonii, Hartig larvae living on Larix lari- L (Hymenoptera, Diprionidae), a wide- cinia, Du Roi (K Koch) (Wagner et al, spread pine sawfly living in European 1983). All these authors concluded that coniferous forests, is able to cause resin acids could be the compounds af- serious damage to Scots pines during fecting sawfly larval development. its outbreaks. Thus, thousands of hec- If abietane and pimarane diterpere tares can be defoliated in less than acids interfere with larval mortality and 2 years (Dusaussoy and Geri, 1966; feeding behaviour in D pini, the quali- Eichhorn, 1982; Geri et al, 1982; Geri tative and quantitative evolutions of and Goussard, 1984; Geri, 1988). these compounds in the foliage should D pini does not feed on young, but be correlated to the habitual feeding on mature, foliage, as is the case for pattern of this insect. many Diprionidae and even other Previous analyses of Pinus sylvestris sawfly species (All and Benjamin, needle resin acids reported the pre- 1975a, b; All et al, 1975; Ikeda et al, sence of labdane diterpene acids such 1977a, b. Wagner et al, 1979; Niemela as manoyl oxid acid (Bardyshev et al, et al, 1982). As previously shown, 1981), pinificolic acid (Enzell and young foliage has a deterrent effect on Theander, 1962), dehydropinifolic acid and affects larval survival of D (Norin et al, 1971, 1980), 4-epi-imbri- pini (Geri et al, 1985, 1988). cataloic acid (Tobolski and Zinkel, Previous results suggested that resin 1982), and of classical pimarane and acids could be involved in the de- abietane diterpene acids such as pi- terrence of young foliage. Thus, maric, isopimaric, sandaracopimaric, abietane and pimarane diterpene acids abietic, palustric, levopimaric, dehy- were tested for antifeedant activity droabietic and neoabietic acids (Norin, against the larvae of various Neodi- 1972; Tobolski and Zinkel, 1982). This paper attempts to correlate the (Geri et al, 1985, 1987). Systematic temporal distribution of abietane and analyses were caried out on selected pimarane diterpene acids in extracts Scots pine foliage during a period from Scots pine needles with the ability ranging from June to September. of D pini to feed on the pine foliage. D pini lives preferably on old trees, Abietane and pimarane diterpene acids the young pines being attacked only were selected because they were usu- during outbreaks (Geri and Goussard, ally tested in sawfly feeding bioassays 1984; Geri, 1988). The same phenom- (Wagner et al, 1983; Schuh and Ben- enon was observed in Sweden on jamin, 1984a, b). Scots pine defoliated by Neodiprion sertifer, Geoff. (Larsson and Tenow, As a of these SOME BIOLOGICAL DATA 1984). consequence pre- vious observations, analyses were also carried out on the of trees of In France, attacks foliage Diprion pini chiefly different Pinus sylvestris but Pinus nigra Arnold ages. Furthermore, resistance ssp. laricio is also weakly damaged at long lasting the end of such outbreaks. P pinaster induced by defoliation (Haukioja and could Aiton is almost never attacked. Exotic Hakala, 1975; Haukioja, 1980) a crucial role in the of species such as P contorta Dougl and play collapse leaf feeder For P radiata D Don show some damage populations. example, (Geri, 1988). Diterpene acid analysis needle quality of Larix decidua re- mained low for defoliators 4 after was carried out on the mature foliage years a decrease in the of these species to find out if the dam- defoliation, inducing success of the insect dini- age could be correlated to pimarane Zeiraphera and abietane resin acid rates. ana Guenée (Benz, 1974; Baltensweiler et Fischlin and Baltensweiler, pini is usually bivoltine in the al, 1977; Diprion Quantitative resin acid Paris Basin. The first generation 1979). changes were shown to occur after wounds in develops from April to July. Eggs are Pinus sylvestris bark (Gref and Erics- laid as as mid-April and hatch be- early We found that the tween late May and early June. The son, 1984). develop- ment of D pini larvae feeding on new larvae feed on the foliage of the pre- of pines defoliated the previous vious years and their growth generally foliage was altered. We observed in ends at the beginning of July. At this year par- ticular a decrease in female time the larvae disperse and transform significant fecondity (Geri et al, 1988). Newly to which spin cocoons on eonymphs, formed Scots needles from trees or in the duff, and then pine vegetation defoliated either in the change successively into pronymphs, artificially pre- vious or by Diprion pini in the pupae, and adults. Adults emerge from Spring Summer, were also extracted the cocoons at the end of July and give previous and birth to the second generation. Larvae analysed. of this second generation develop be- tween late August and October. These MATERIALS AND METHODS larvae feed on previous year as well as current year foliage. Thus as shown by the new of previous bioassays, foliage Current year needle samples were collected Scots pine is repellent to D Pini larvae in 1984 on June 27th, July 16th, August 1st, and this repellency decreases with time 14th and September 10th on several twigs of the same 5 ca. 10 year-old pines from a The resin acids were converted by dia- pine plantation at Olivet, INRA Forest Labo- zomethane to the corresponding methyl ratory, near Orléans (France). This plantation esters and analysed by Gas Chromatogra- is an homogenous plantation growing from phy (Varian series 1 400, Flame lonisation wild seeds (the most likely origin being from Detector) on an Alltech RSL 150 Megabore Hagueneau forest). All the trees of this plan- column (15 m x 0.53 mm). The oven tation were normally attacked by Diprions temperature was programmed between 120 during the last outbreak but were free of °C and 180 °C at 6 °C/mn, from 180 °C to sawflies for at least three years. Their foliage 195 °C at 1 °C/mn and from 195 °C to 255 was used for feeding and breeding experi- °C at 2 °C/mn. Individual resin acids were ments which have been reported in other pa- identified by direct comparison and cochro- pers (Geri, 1986; Geri et al, 1988). On May matography with authentic samples (Helix 22th, 1984, samples of foliage formed in Biotech. Ltd, Vancouver, Canada) and by 1H 1982 and 1983 were also collected. N M R after NO3Ag TLC isolation. Absolute amounts of resin acids were estimated The role of tree age was studied using by 5, 10 and 30 year-old pines (from the Olivet peak area triangulation, compared with resin plantation for the first 2 and from the Orléans acid standard solutions and corrected by reference to an internal standard of forest for the latter) from which one year-old methyl- The data are the aver- needles were collected on May 28th and palmitate. reported of at least 3 different determinations June 6th, 1984. The defoliation effect was ages carried out on the same material. investigated on 10 year-old pines (Olivet pine plantation) which were partly defoliated by man during the spring of 1983 or by D pini larvae during the Summer of 1983. The RESULTS current year foliage from artificially defoliated pine was collected on July 19th, 1984 and from the defoliated naturally pine Lipids, acid fractions, abietane and pi- on September 10th, 1984. marane diterpene acid rates in Scots The collected needles were frozen and in relation to the needle kept at -20 °C until extraction and analysis. pine foliage I. in- Fifty g (fresh weight) of each sample were age, are listed in table Lipids ground using a Waring Blendor and ex- crease in the current year foliage tracted 3 times with methanol/dichloro- during the growing season (from 2.45% methane 1/1 (v/v). The solutions were filtered to 7.1 % dw). The acid fraction contain- a filter and the crude through glass fritted ing the pimarane and abietane resin extracts were dried in a Bûchi rotavapor at a evolution from ambient temperature. The needle dry weight acids follows parallel (dw) is the sum of the crude extract and ex- 0.84% to 2.03% (dw). These acids are tracted needle dry weights. principally represented by pimarane The crude extract was fractionated into acids (mainly sandaracopimaric acid) acid and neutral fractions by agitation with while the abietane acids (except for the 2% NaOH, followed by dichloromethane ex- 08/01/84 sample) represent only 25%- traction. After the neutral organic phase 50% of their rates. The total level of elimination, the NaOH phase was aqueous acids increases from acidified with 1.2 N HCl and the acids ex- these early Spring tracted with dichloromethane. to late Summer (from 0.030 ‰ to 0.130 ‰ the different The acid fraction was chromatographed dw). However, by reverse phase HPLC (Perkin Elmer 2A groups do not show the same evolu- pump with LC 75 UV-Visible detector at tion: pimarane resin acids gradually in- 241 nm) on a Whatman Partisil M9 10-50 C8 crease during the growing season in x 9.4 The elution mixture column (500 nm). contrast to abietane resin acids which was methanol/water/isopropanol/orthophos- maintain the same level phoric acid 350/150/50/0,1 (v/v) at 3 ml/mn. throughout (ex- The pimarane and abietane diterpene acids cept in August, which had a higher flowed out together after 50 min. value). We did not manage to detect any trace of levopimaric, or palustric The abietane and pimarane diter- acids. In early Spring, 1 or 2 year-old pene acid contents determined in the foliage, as well as current year foliage, extracts of 5, 10, and 30 year old pine contained similar amounts of resin foliage are reported in table II. Old acids (between 0.012 ‰ and 0.03 ‰ pines (10 and 30 year-old) contain dw). higher levels of diterpene acids in their There is no obvious connection be- foliage and an evolution occurs with resin acids tween a low rate of pimarane and time, the pimarane being abietane diterpene acids and the ability more abundant in 10 year-old speci- resin acids becom- of D pini to feed on pine foliage as mens and abietane in The shown by observations in nature or with ing more abundant old pines. laboratory experiments as summarised oldest Scots pine are more easily at- in table I (these observations were re- tacked by Diprions than the youngest, to ported in other papers - Geri et al, so that the feeding ability appears 1986, 1987). In Spring these diterpene be correlated with a high level of diter- Such for acid rates are low both in the previous pene resin acids. preferences were noticed Geri and year foliage which is not antifeedant older trees by for P and and in the new foliage which is an- Goussard (1984) sylvestris attacked tifeedant, while they are high in late also observed for pines by Summer and Autumn in the current year Neodiprion sertifer Geoffr in Southern and foliage which can be consumed by the and Central Sweden (Larson Diprions. Nevertheless, in the 1 or 2 Tenow, 1984). year-old pine foliage, these rates are The abietane and pimarane diter- particularly low with regard to the total pene acid rates determined in the new lipid fraction. foliage of P sylvestris after a previous defoliation are listed in table III. These and September samples respectively). results show an increase in the total Nevertheless, the average of abietane lipids compared with normal foliage. In and pimarane diterpene acids relative fact, this phenomenon is due to a high to the needle dry weight is also about increase in the neutral lipids which twice as high in previously defoliated doubled (from 2.18% to 5.14% and new foliage collected in July and Sep- from 5.07% to 10.93% dw for the July tember than in undefoliated pine in the other needles from the same periods. This in- with the observed rates for ex- crease seems to be mainly due to species (73.5 in P pinaster abietic and neobietic acids while pi- ample). marane diterpene acids decreased slightly in the September experiment. Levopimaric and palustric acids were DISCUSSION also observed in these lipid fractions as shown in table III. The acid fraction level (0.76% to 2.03% Abietane and pimarane diterpene dw) found in Scots pine needles is acid rates of the main French pine spe- about the same as that found by Enzell cies on which D pini can develop more and Theander, 1962, (0,043% dw), or or less easily are given in table IV. The by Norin et al, 1971, (1,86% dw). How- ability of this insect to live on a partic- ever, pimarane and abietane diterpene ular pine species is hard to correlate acids represent only a small part of this with the presence (or absence) of any fraction (about 0.1 % to 1 % of these characteristic resin acid. All species acids). have about the same acid fraction Tobolski and Zinkel, 1982, found a level, namely 0.05 to 0.12% of foliage higher resin acid rate which evolved dw. However, Scots pine foliage con- from 33.4 mg/g to 45.7 mg/g dw tains less abietane and pimarane diter- (namely 3.3% to 4.57% dw) in the ex- pene acids than other species (20 ppm tracts of Scots pine needles. In their reported for P sylvestris, 112 ppm for studies, pimarane and abietane diter- and 55 for P pinaster and 42, 47, ppm pene acids represented from 13%-40% P radiata, P contorta and P laricio re- of the total resin acids. Thus they, re- spectively). Moreover, we observed that ported values which are 10-40 times acid level the Scots pine abietane resin higher than our data. It is difficult to un- is particularly low (3.8 ppm) compared derstand the difference between pre- vious observations and the present re- and Ericson, 1984), or wood analyses sults. One could, perhaps, explain - about 0.74% dw - (Yildrim and Holm- these discrepancies by genetic diver- bon, 1977). These results cannot be gences between North American, North compared with our data since we have European and Central European spe- only analysed the needles. Like Greff cies. Larsson et al, 1984, stated that and Ericsson, 1984 and Gref and the resin acid rates could be charac- Tenow, 1987, we observed an increase teristic of some clones; they reported of pimarane and abietane diterpene the existence of clones with high levels acids during the growing season while of resin acids (5.2% dw) and of others Tobolski and Zinkel, 1982 found an op- poorer in resin acids (1.52% dw). The posite pattern. first contained twice as much abietic, In studies on the predominant role levopimaric, and palustric acids than of resin acids in the control of sawfly the latter, but, unfortunately, these populations, different authors (Ohigashi authors did not data on the give any et al, 1981; Wagner et al, 1983; Shuh levels of pimarane diterpene acids. Cli- and Benjamin, 1984a, b) reported that matic factors such as humidity, abietane and pimarane resin acids temperature, or sunlight are not negli- added to mature foliage inhibited larval gible; indeed, recently, Gref and Tenow, feeding and growth. They concluded 1987, that needles from reported sunny that these resin acids may contribute sites contained more resin acids than significantly to the natural deterrence of needles from the shade dw for (2.24% the current season foliage against Di- the first and 1.37% dw for the second); prions. Their conclusions are drawn in this as in that of the study previous from experimental results and a pre- authors, the level of pimarane diterpene vious observation by Ikeda et al, 1977, acids is not mentioned. who found in P banksiana foliage a Moreover, all these authors worked diterpene acid (13-keto 8(14)-podocar- on an acidic fraction which contained pene 18-oic acid) which deterred larval compounds such as labdane diterpene feeding of N rugifrons and N swanei. acids and their oxidised derivatives in This compound occurred at high levels addition to the pimarane and abietane in Spring in the new foliage and diterpene acids. We have shown that decreased throughout the growing sea- pimarane and abietane resin acids rep- son. resent only a small part of the total acid With D pini, the nutritional experi- fraction, of labdanic mainly composed ments that we made, gave doubtful re- acids and of derivatives hydroxylated sults (Geri et al, 1985). In addition we of acids et diterpene (Buratti al, 1987). found an increase of the amount of 13- In our study, the isolation of the total keto 8(14)-podocarpene 18-oic acid in acids HPLC allows us to obtain by the P sylvestris foliage during the grow- abietane and acids pimarane diterpene ing season (Buratti et al, 1988). Now, the more together; polar hydroxylated we have shown that the pimarane and diterpene acids such as the labdane abietane diterpene acids increase from acids, are the diterpene separated by early Spring to Autumn. That is to say same chromatography. that the increase of the supposed de- Other data concerning P sylvestris terrents correspond with the feeding result from pine seedling bark analyses season of D pini larvae - a rather con- - between 0.8% and 3% dw - (Gref tradictory statement. After these obser- vations it is difficult to conclude that tacked pine contains the highest rate these acids have a determinant role in of abietane diterpene acids. the choice of foliage during Diprion at- From all these results, we can con- tacks. If such a relationship existed, it clude that, if the abietane and pi- would be advisable to observe the cor- marane diterpene acids of Scots pine relation between high levels of these needles can interfere in the D pini compounds in the pine needles and the development, they probably cannot be incapacity of D pini to live on them. considered as determinant factors for of this It We observed that D pini reared on the natural equilibria species. Scots pine, defoliated the previous is noticeable however that pimarane acids increase year, developed more quickly resulting diterpene regularly in less weight gain and reduced fecun- during the growing season, an evolu- dity (Geri et al, 1988). Niemela et al tion which is modified little by previous 1984, reported that Neodiprion sertifer defoliations while the amount of abietane acids is developed more quickly on pines which diterpene greatly modified defoliations. were partly defoliated the previous by Summer while Microdiprion pallipes showed a higher mortality. There is no doubt that defoliation can have an in- REFERENCES fluence on the defoliators which develop on the pines the following year. 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