*Corresponding author. Tel.:#58-2-6051307; fax:#58-2-6051204. E-mail address:[email protected] (A. Ceden8o).
Inter-population di
!
erences in the essential oils
of
Pinus caribaea
needles
Luis Felipe Barnola
!
, Aragua Ceden
8
o
"
,
*
!Apartado 47058, Caracas 1041-A, Caracas, Venezuela"Facultad de Ciencias, Instituto de Zoologn&a Tropical, Universidad Central de Venezuela, Apartado 47058, Caracas 1041-A, Venezuela
Received 10 March 1999; accepted 18 October 1999
Abstract
Di!erences in volatile terpene content ofPinus caribaeaneedles and soil qualities between
two pine plantations (Uverito and Sartenejas) in Venezuela were analyzed. Soils in the Uverito
pine plantation were sandy, low in nutritional quality, and de"cient in available nitrogen and
phosphorus content. Pines grown on these soils presented in their needles higher concentrations
ofa-pinene, camphene, sabinene, myrcene#a-phellandrene mixture,b-phellandrene,b
-cary-ophyllene,a-humulene, and a higher total monoterpene content than pines of the Sartenejas
plantation, where soils were nutritionally richer and higher in nitrogen and phosphorus content. The hypothesis of the carbon/nutrient balance could explain these results. Alterna-tively, continuous stress on the pines of the Uverito plantation due to herbivory by the
leafcutter antAtta laevigatamay induce, in part, the di!erences observed between these pines
and those of the Sartenejas plantation. ( 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Pinus caribaea; Monoterpene; Sesquiterpene; Inter-population di!erences; Carbon/nutrient balance hypothesis
1. Introduction
Intraspeci"c variations in volatile terpene content in leaves of certain conifers
related to the geographical distribution of the species have been reported (Rudlo!and
Rehfeldt, 1980; Hall and Langenheim, 1987; SchoKnwitz et al., 1990; Ra"i et al., 1992),
in particular of the genusPinus(Zavarin et al., 1976; Snajberk et al., 1978; Cool and Zavarin, 1992; Nerg et al., 1994). Numerous taxonomic studies are based on the variability of mono- and sesquiterpene content in various conifers (Zavarin et al., 1976; Snajberk et al., 1978; Rudlo!and Rehfeldt, 1980; Ra"i et al., 1992).
Variability in volatile terpene content in leaves of certain conifers may play an important role in their interactions with herbivorous insects (Edmunds and Alstad, 1978; Gambliel and Cates, 1995; Zou and Cates, 1995). Geographical variability in
terpene content of the oleoresin ofPinus ponderosa has been related to di!erential
selection pressures by specialist herbivores (Sturgeon, 1979; Sturgeon and Mitton, 1986).
Environmental factors such as water and nutrient (nitrogen, phosphorus)
availabil-ity may in#uence the essential oil production in leaves of several plant species
(Mihaliak and Lincoln, 1985, 1989; Muzika et al., 1989; Ross and Sombrero, 1991; Kainulainen et al., 1992; Yani et al., 1993). Variations in soil quality and fertilization
induce di!erences in leaf secondary metabolite chemistry of Inga oerstedianawhich
may in#uence the herbivory patterns of the leafcutter ant Atta cephalotes
(Nichols-Orians, 1991). Changes in leaf primary and secondary metabolite chemistry have been
related to defoliation inP. ponderosa(Wagner and Evans, 1985).
Variability in terpene content inPinus caribaearesin has been studied (Green et al., 1974, 1975), including taxonomic aspects of the several varieties (Coppen et al., 1993). However, little is known with regard to terpene content in needles of this species (Ekundayo, 1978) and its intraspeci"c variability (Barnola et al., 1994, 1997). The
selective herbivory of the leafcutter antAtta laevigata in a plantation of Caribbean
pine was found to be related to terpene variation in pine needles (Barnola et al., 1994).
Pinus caribaeawas introduced to Venezuela around 1969. One plantation has been established in Uverito, Monagas State, and large areas in the mountains of Sartenejas,
Miranda State, have been planted with this pine species. Both the localities di!er in
climatic conditions and abiotic factors such as soil characteristics. High population densities ofAtta laevigata, which cause signi"cant economic damage, were observed in the Uverito plantation. This ant species is also found in small numbers in the
Sartenejas woods, where LeoHn (1986) observed low herbivory intensity on Pinus
caribaea. As part of a long-term study on the relationship betweenPinus caribaeaand the herbivory byAtta laevigata(Ceden8o, 1989; Barnola et al., 1994, 1997), we investi-gated possible inter-relations between abiotic factors, essential oil chemistry and ant herbivory in these two locations.
2. Materials and methods
2.1. Locations
The Uverito plantation is located in a savanna in Monagas State (8335@N, 62350@
W) at a mean altitude of 60 m, with an annual mean temperature of 26.33C and annual
Miranda State (10325@N, 66353@W) are found on hilly terrain at a mean altitude of
1300 m, with an annual mean temperature of 19.43C and annual mean precipitation of
983 mm. Greatest precipitation is between April and November followed by a dry season from December to March.
2.2. Plant material and sampling procedure
Samples of mature needles were taken from seven Caribbean pines (Pinus caribaea
Morelet var. hondurensis Barrett and Golfari) at the Uverito plantation and 13
Caribbean pines from the Sartenejas woods. All the trees were over 15 m hight. In order to reduce variability in terpene content due to di!erences related to canopy level and diurnal and seasonal changes (Barnola et al., 1997), pine needle samples were
taken from the four cardinal directions from the lower canopy region ((5 m) from
each pine in both localities in the morning during the dry season (March}April, 1994). Collections from branches within a tree were combined and immediately immersed in liquid nitrogen until laboratory processing.
2.3. Terpene analysis
Every pine needle sample was powdered in a homogenizer (Omnimixer) containing liquid nitrogen. Two-hundred milligrams fresh weight of frozen needle powder were
extracted in the dark with 5 ml HPLC-graden-pentane at #43C for 96 h. Ten ll
n-tetradecane solution inn-heptane (140ll 25 ml~1) were added to each extract as an internal standard. Each extract was"ltered and concentrated to approximately 600ll under a mild stream of nitrogen and immediately analyzed by GLC. Seven microliters
of concentrated extract were injected into the GLC column [5% AT}1000 on
Chromosorb WHP 80}100 mesh, 5 m]4 mm i.d. glass column, 45 ml min~1 He
carrier gas, column temperature: 503C (16 min) to 2003C at 23C min~1, injector at
1503C, FID at 2003C]. Peak areas were measured with an electronic integrator and
quanti"ed with respect of the internal standard (n-tetradecane). Relative response factors to n-tetradecane were determined using several mono- and sesquiterpene
authentic samples. Compound identi"cations were achieved by comparing retention
times with those of several mono- and sesquiterpene standards and by determining their KovaHts'indices (Davies, 1990). Identities were con"rmed by comparing relative retention times with those obtained in a previous study (Barnola et al., 1994).
2.4. Soil analysis
Seven soil samples were taken (10}40 cm depth) from the Sartenejas woods and 17
Fig. 1. Graphic representation of camphene and sabinene concentrations (lg g~1) in pine needle samples
from Sartenejas and Uverito plantation. 2.5. Statistics
A Mann}Whitney U-test was done between Sartenejas and Uverito plantation on
data corresponding to terpene concentrations and soil characteristics. The most signi"cant variables were graphically represented in Figs. 1 and 2. STATISTICA 5.0 program was used for the statistical analyses.
3. Results
Individual terpene concentrations and total monoterpene content (in lg g~1 of
fresh needle) in pine needles from Uverito and Sartenejas are shown in Table 1. Major
components were b-phellandrene (8), germacrene D (16), b-caryophyllene (13),
limonene (7),a-pinene (1). According to results of the Mann}Whitney U-test in Table
1, the concentrations ofa-pinene (1), camphene (2), sabinene (4), myrcene#a
-phellan-drene mixture (6),b-phellandrene (8),b-caryophyllene (13), anda-humulene (14) were signi"cantly higher, and that of germacrene D (16) signi"cantly lower in the pine needles of Uverito compared to those in Sartenejas pine samples. Fig. 1 shows camphene and sabinene concentrations in pine needle samples from Sartenejas and Uverito plantation. These variables were the most signi"cantly di!erent between the two locations (Table 1).
The averages of variables in soil samples from Sartenejas and Uverito plantation
are shown in Table 2. According to the Mann}Whitney U-test in Table 2, all
Fig. 2. Graphic representation of nitrogen and phosphorus concentrations (ppm) in soil samples from Sartenejas and Uverito plantation.
and sand percentage, were signi"cantly higher in the soil samples from Sartenejas
compared to those for the soil samples from Uverito plantation; and sand was signi"cantly higher in the soil samples from Uverito plantation compared to that from Sartenejas. Fig. 2 is a graphic representation of nitrogen and phosphorus concentra-tions in soil samples from Sartenejas and Uverito plantation.
4. Discussion
Results shown in Table 1 regarding the qualitative composition of essential oils in Caribbean pine needles are similar to those found in previous studies (Barnola et al., 1994, 1997). Also, the values of the relative silt#clay content, as well as pH values,
found for Uverito soils (Table 2) agree with the values reported by other researchers (Fassbender et al., 1979). In general, the nutritive elements found in Uverito soils
re#ect a low fertility and limited nutritional quality, a conclusion also shared by
Fassbender et al. (1979).
These results indicate that there are important di!erences between the Uverito and
Sartenejas localities in soil characteristics and needle composition of volatile terpenes. Mean concentrations of most terpenes are higher in pine needles of Uverito than those
in pine needles of Sartenejas (Table 1). Therefore, the low herbivory intensity byAtta
Table 1
Identity and mean concentration (lg g~1needle fresh wt) of mono- and sesquiterpenes in Caribbean pine
needles from pine plantations at Uverito and Sartenejas Mean concentration (lg g~1)
Compound Uverito (n"7) Sartenejas (n"13)
Mean S.E. Mean S.E.
6 Myrcene#a-Phellandrene! 124 14 90 10 0.043
7 Limonene 66 43 204 80 0.278
8b-Phellandrene 1030 132 600 74 0.016
9c-Terpinene 9 4 4 1 0.475
10a-Ocimene 7 7 12 8 0.435
11 Unidenti"ed 4 1 3 1 0.842
12 Bornyl acetate 24 6 29 8 0.842
13b-Caryophyllene 366 78 192 39 0.029
14a-Humulene 64 14 31 7 0.017
15c-Muurolene 17 3 15 5 0.153
16 Germacrene D 343 33 659 141 0.029
17b-Cadinene#c-Cadinene! 33 7 25 4 0.362
Total
Monoterpenes 1503 142 1099 125 0.036
!Peaks not resolved satisfactorily.
are, in general, limited in nutritional quality compared to those of Sartenejas (Table 2). These results are consistent with those obtained by others who reported higher total volatile terpenoid concentrations in leaves of individuals of several plant species growing in low nitrogen and phosphorus availability (Mihaliak and Lincoln, 1985; Mihaliak and Lincoln, 1989; Muzika et al., 1989; Ross and Sombrero, 1991).
Nitrogen and phosphorus availability for the pines of Uverito is drastically lower (as shown in Table 2 and Fig. 2) compared to that of the pines of Sartenejas, whereas
the concentrations of several terpenes are signi"cantly higher in pine needles of
Uverito compared to those in pine needles of Sartenejas (Table 1). These facts seem to corroborate the carbon/nutrient balance hypothesis (Bryant et al., 1983), according to which, in restricted growth conditions limited by nutrient availability, part of the
carbon"xed by the plant is involved in the biosynthesis of secondary metabolites,
Table 2
Mean values for soil characteristics from Sartenejas and Uverito plantation Sartenejas
Organic matter (%) 1.6 0.6 0.7 0.1 0.13549
pH (in water, 1 : 1 w/v) 4.6 0.1 4.5 0.1 0.20372
Total nitrogen (ppm) 1330 177 112 25 0.00012
P (as PO3~4 ) (ppm) 2.0 0.2 0.6 0.1 0.00016
K (ppm) 28 5 6 1 0.00016
Ca (ppm) 121 28 36 8 0.01011
monoterpenes) is signi"cantly higher in pine needles of Uverito compared to that in pine needles of Sartenejas (Table 1).
Some terpenes, likeb-caryophyllene and myrcene#a-phellandrene mixture, found
in higher concentrations in the pine needles of Uverito compared to those found in Sartenejas pine samples, may play an important role in the interactions with the
leafcutter ant Atta laevigata in the Uverito pine plantation (Barnola et al., 1994).
Results obtained in this former study seem to indicate that the herbivory byAtta
laevigatamay induce changes in terpene content in the needles of defoliated Cari-bbean pines of the Uverito plantation, and these changes are characterized by an increase in the concentration of certain terpenes in the pine needles. On this basis, we speculate that the higher concentrations of several terpenes found in this study in pine
needles of Uverito (compared to those in pine needle of Sartenejas) re#ect a stressed
condition in these pines, which may be due to the low nutritional quality of the
Uverito soils, and, probably as importantly, to the constant pressure exerted byAtta
laevigataherbivory on these trees, since the population density of this ant is consider-ably higher in the Uverito plantation compared to that observed in Sartenejas (personal observations). However, these hypotheses need further testing.
Acknowledgements
We are grateful to Jorge Zegarra, NapoleoHn LeoHn, AdriaHn LeoHn, Cristian Grases,
assistance, and to Eligio Oropeza and Masahisa Hasegawa for laboratory assistance.
Ismael HernaHndez advised on soil data interpretation and Luis Bulla on statistical
analyses. Alejandro AmormHn and VmHctor Hugo Aguilar kindly supplied some materials.
We appreciate critical comments on the manuscript by Luisa Elena Cardozo, CeHsar
Ovalles, Oscar Barnola, Zoila GonzaHlez, Jorge Zegarra, and two anonymous
re-viewers. CVG-PROFORCA furnished food and lodging in the "eld. Funds were
provided by Consejo de Desarrollo CientmH "co y HumanmHstico de la Universidad
Central de Venezuela (03-313261-94) and CONICIT (S1-2257).
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