5.4 PLANT COMPOUNDS AND RESIN IN RESISTANCE MECHANISMS

5.4 PLANT COMPOUNDS AND RESIN IN RESISTANCE MECHANISMS

Resistance of pines to bark beetle attacks has long been attributed to the amount of resin exudation and formation of pitch tubes (Webb, 1906). It is obvious that oleoresin acts to entrap and impede the excavation efforts of bark beetles. Dead beetles can often be seen in crystallized resin of pitch tubes. Fig. 7. Western pine beetles, Dendroctonus brevicomis, "swimming" in oleoresin exuding from a "pitch tube" on the bark of ponderosa pine. The beetle (in center of photo) and its mate underneath were observed to burrow down through the viscous liquid into the entrance of the gallery (directly under and to right of beetles) for up to several minutes before returning and shoving out more resin. The resin is slightly toxic and may exhaust the beetles; it also may eventually crystallize to entrap them.

However, D. brevicomis and other aggressive bark beetles have a great ability to survive the "toxic" monoterpenes and suffocating mucilage and may struggle for hours in copious resin flows (D. frontalis, Hodges et al., 1979). I have observed D. brevicomis beetles completely covered with resin while attempting to clear the entrance tunnel (Fig. 7), and it appears that "breaths" of air are taken by slightly lifting the elytra that protect the spiracle openings on the dorsal side of the abdomen; later these beetles entered the tree and made galleries (Byers et al., 1984). In experiments where three female D. ponderosae were caged on each of 79 trees, only 43 trees were attacked, and of these just 15 were colonized by aggregating beetles (Raffa and Berryman, 1979).

Oleoresin may provide resistance to trees due to chemical toxicity to the beetle and associated microorganisms or to physical impedance and entrapment (Hodges et al., 1985). Oleoresin and the monoterpenes therein are repellent to bark beetles in concentrated amounts (Struble, 1957; Pitman et al., 1966; Berryman and Ashraf, 1970; Bordasch and Berryman, 1977). Drought and poor water balance lowers the resistance of conifers (Hodges and Lorio, 1975; Hodges et al., 1979) probably by lowering the turgidity of resin duct cells which lowers the oleoresin exudation pressure (OEP). A correlation between higher OEP and greater resistance of ponderosa pine to attack by D. brevicomis and I. paraconfusus has been reported (Vité, 1961; Wood and Vité, 1961; Wood, 1962; Brown et al., 1987). Hodges et al. (1979) found that in more resistant pines their resin was slower to crystalize (P. elliotii) or had a higher resin flow (P. palustris) compared to more susceptible trees (P. taeda and P. echinata) colonized by D. frontalis. Cook and Hain (1987) also found that susceptible shortleaf pines, P. echinata, had a lower resin flow than resistant trees. However, Raffa and Berryman (1982b) found no relationship between resistance and the rate of resin flow or crystallization. Also, Schroeder (1990) found no difference in resin flow between resistant and susceptible Scots pine, P. sylvestris, feed on by T. piniperda. Western larch, Larix occidentalis, had no OEP but the trees with higher content of 3- carene in the resin were attacked less by D. pseudotsugae (Reed et al., 1986).

Another factor that might be more important for resistance could be the toxicity of compounds within the resin. Smith (1961, 1965a, b) exposed beetles of several Dendroctonus species to resin vapors from host and nonhost pines and found beetles were able to tolerate vapors of their host better than nonhosts. In an attempt to determine which components of the resin vapor were toxic to bark beetles, various conifer monoterpenes were presented neat at vapor saturation to D. brevicomis held in a glass chamber (Smith, 1965a). The most toxic monoterpene was limonene, followed by (+)-3-carene, myrcene, (-)-B-pinene and then alpha-pinene. In another study, Smith (1965b) found that n-heptane, a major constituent of Jeffrey pine (P. jeffreyi), was quite toxic to D. ponderosae which does not feed in this tree, whereas Dendroctonus jeffreyi beetles showed little mortality in saturated vapors. However, the monoterpenes when presented alone at saturation were from 40 to 80 times higher in concentration than in the natural atmosphere of a beetle's gallery, as found empirically by GC headspace analysis and explained by Raoult's law that states the vapor pressure of a compound is due to its mole percentage in the substrate/solvent mixture (Byers, 1981a). Most of these species may also avoid monoterpene vapors temporarily by breathing at "ventilation" holes through the outer bark. Thus, Smith's conclusion that monoterpenes are toxic under natural conditions is dubious, but his results may still indicate these monoterpenes increase mortality of bark beetles over longer periods during feeding and colonization. Raffa et al. (1985) exposed Scolytus ventralis to the above monoterpene vapors and also found limonene the most toxic, but myrcene, alpha-pinene, B-pinene, and 3-carene, in that order also caused significant mortality.

Differences in monoterpene composition within a tree are slight, even from year to year (Smith, 1964; Byers and Birgersson, 1990). Differences in monoterpene ratios can be large between trees of one area, almost as large as differences over wide geographic regions (Smith, 1964, 1966, 1967, 1969). The oleoresins of 88 trees under attack by D. brevicomis and subsequently killed were compared to those from 202 living trees, and the living trees had a higher content of myrcene and limonene in their resins (Smith, 1966). This correlation supports the theory that limonene is important in host resistance.

Sturgeon (1979) theorized that D. brevicomis is a selective force in the evolution of P. ponderosa and that therefore areas of recent outbreaks might have selected trees with a higher titer of limonene (since these trees would be more resistant). Eight populations of trees, a total of 617 trees, were sampled for monoterpenes and analyzed by principal component multivariate statistic. The average proportions of myrcene and alpha-pinene in resins from the eight populations ranged the least (10.0-15.2% and 4.5-9.2%, respectively), while the other monoterpenes varied more (3-carene, 23.6-60.4%; B-pinene, 13-35%; limonene, 6.1-18.7%). The populations were separated by the Cascade Range in northern California and southern Oregon into two regions. The west side had higher proportions of myrcene, B-pinene, and limonene. The proportion of limonene in resin from three populations considered to have been historically under heavy bark beetle predation was higher than in populations not considered to have such a history (Sturgeon, 1979). The problem with these findings is that a correlation is made between rather accurate monoterpene results and rather poor knowledge of former predation pressure by D. brevicomis, or for that matter other bark beetle species in each of the areas.

Sturgeon (1979) concluded that D. brevicomis beetles (and others) may exert a frequency-dependent selection pressure on chemically polymorphic populations of ponderosa pine. Thus after beetles have colonized most of the low limonene and less resistant trees during an epidemic, the beetle population would either (1) die out or disperse to areas that had more chemically susceptible trees or (2) evolve a tolerance to limonene. The second possibility was considered less likely because of the large variation in monoterpene composition among trees that would make it improbable that selection of beetles would occur that were capable of detoxifying all of these compounds. However, bark beetles, including I. paraconfusus and D. brevicomis, already must be able to detoxify all of these compounds since they survive exposure to monoterpene vapors in part by converting them to oxygenated products that are more soluble and readily excreted (Hughes 1973, 1974; Byers, 1981a, b, 1982, 1983b, c; Pierce et al., 1987). Another hypothesis is that limonene might not always be the most toxic to a bark beetle population, but rather those monoterpenes that the population is not well adapted to (since they occur infrequently) are the most toxic. In this regard, lodgepole pines in some regions of California have very high titers of limonene (Byers and Birgersson, 1990), yet they are readily attacked and killed by D. ponderosae.

Gollob (1980) measured the monoterpene content of resin from unattacked loblolly pines, P. taeda. Two apparently resistant pines that had survived attack by D. frontalis in an epidemic area had a much higher content of myrcene compared to other trees that were killed by the beetle and had low or trace amounts of myrcene. However, no consistent differences in monoterpene composition of Douglas-fir, P. menziesii var. glauca, resin were found between trees that had resisted attack by D. pseudotsugae and trees that had succumbed (Hanover and Furniss, 1966). Similarly, Raffa and Berryman (1982b) found no relationship between monoterpene composition and degree of resistance of lodgepole pines, P. contorta var. Latifolia, to D. ponderosae. Hodges et al. (1979) also did not find differences in monoterpene or resin acid composition which could account for differences in resistance among four pine species to attack by D. frontalis.

In addition to the wound or primary resin production, conifers have a secondary or hypersensitive response to attack (Reid et al., 1967; Berryman 1969, 1972; Berryman and Ashraf, 1970; Raffa and Berryman, 1982c, 1983; Christiansen et al., 1987). The tree responds by isolating the invading insect or fungus within a lesion of dead cells and secondary resin by autolysis of cells and formation of traumatic resin containing higher concentrations of monoterpenes and phenolics (Reid et al., 1967, Berryman, 1969, 1972, Shrimpton, 1973, Wong and Berryman, 1977; Wright et al., 1979; Raffa and Berryman, 1982b, c, 1983; Hain et al., 1983, Croteau et al., 1987).

Croteau et al. (1987) identified elevated levels of monoterpenes and diterpene resin acids in stems of lodgepole pine inoculated with blue-stain fungus Ceratocystis clavigera, resulting in induced lesions and secondary resin production. The inoculated stems contained about three times more of the monoterpenes alpha- pinene, B-pinene, 3-carene and B-phellandrene, but less limonene. De novo resin synthesis was indicated in the infected tissue since radiolabelled sucrose was incorporated up to 20 times faster into monoterpenes and up to 10 times faster into diterpene resin acids. Chitosan, a fungal wall fragment, induced monoterpene biosynthesis and increased levels of terpene cyclase enzyme which converted radiolabelled terpene precursors (geranyl pyrophosphate and farnesyl pyrophosphate) to labelled monoterpenes and sesquiterpene olefins, respectively (Croteau et al., 1987). Nearly all S. ventralis females leave their entrance holes in grand fir, Abies grandis, when hypersensitive reactions are evident (Berryman and Ashraf, 1970). Shorter egg galleries are made or fewer eggs are laid by females in lesions (Berryman and Ashraf, 1970; Paine and Stephen, 1988).

The hypersensitive wound reaction not only affects bark beetles directly, but inhibits symbiotic microorganisms from growing and killing the tree. Blue-stain and other symbiotic fungi are surrounded and isolated in the lesions (Wong and Berryman, 1977; Stephen and Paine, 1985; Paine and Stephen, 1987, 1988). Sometimes, phytopathogenic fungi (e.g. Ceratocystis minor var. barrasii) that are carried in the beetle's mycangium (D. frontalis) do not stimulate the hypersensitive response and thus spread to kill the tree (Paine and Stephen, 1987). Possibly the fungi secrete compounds, such as water soluble glycans, that inhibit the plant's defensive hypersensitivity - as in potatoes (Doke and Tomiyama, 1980). Cobb et al. (1968) cultured four species of Ceratocystis fungi associated with bark beetles (species: ips, minor, schrenkiana, and pilifera) as well as the root pathogen fungus, Fomes annosus, during exposure to saturated atmospheres of oleoresin or monoterpenes of the host ponderosa pine. They found that all fungal species were inhibited in growth by oleoresin except C. ips, while all the monoterpenes (the five discussed above plus B-phellandrene and camphene) as well as undecane (present in Jeffrey pines) inhibited growth of the fungal species. Camphene and undecane were the least toxic, alpha-pinene was intermediate, while B- pinene, B-phellandrene, and 3-carene were more toxic, with myrcene and limonene the most toxic. However, the most toxic of all compounds tested was n-heptane (also the most volatile), a major constituent of Digger pine, P. sabiniana (Mirov, 1961). When the monoterpenes were incorporated into the culture medium as well as in the vapor phase, myrcene and B-phellandrene appeared the most toxic to most species, but all of the monoterpenes and undecane reduced fungal growth (Cobb et al., 1968). They also indicated that (+)-alpha-pinene was more inhibitory than the (S)-enantiomer. The growth inhibition by monoterpenes was proposed to allow the tree time to synthesize phenols (Shain, 1967) such as pinosylvin (Anderson, 1962) that would kill the fungi (Cobb et al., 1968).

Bridges (1987) tested alpha-pinene and B-pinene on two mycangial fungi of D. frontalis and the blue stain fungi, C. minor. B-pinene inhibited growth of C. minor, both monoterpenes inhibited one mycangial fungi while the other fungus was stimulated. A phenylpropanoid, 4-allylanisole, from P. taeda resin inhibited growth of all fungi tested. Himejima et al. (1992) steam distilled ponderosa pine oleoresin into a distillate of monoterpenes and sesquiterpenes and a residue of four diterpene acids. The individual monoterpenes of the distillate were not inhibitory to growth of several gram-positive bacteria (at 800 æg/ml) but did inhibit two species of fungi. Longifolene, a sesquiterpene, inhibited the gram-positive but not the gram-negative bacteria. Other species of common mold fungi were not affected by the monoterpenes, but the diterpene acid, abietic acid, was effective against three species of gram-positive bacteria. Although these species of microorganisms are not those associated with bark beetles, the results indicate that oleoresin constituents may be important as a general defense against microorganisms.

The carbon balance of a tree is the relative level of photosynthate available for growth, maintenance, and biosynthesis of defensive compounds. Christiansen et al. (1987) have reasoned that too little moisture (drought), insect defoliation, and root pathogens will reduce the amounts of carbon available for biosynthesis of primary and secondary resin. Mild drought may actually increase resistance of trees by lowering growth rates and shifting the use of photosynthate to the biosynthesis of defensive chemicals, while extended drought will increase the probability of bark beetle outbreaks due to the depleted carbon reserves (Christiansen et al., 1987). A similar theory of tree resistance and attack by southern pine beetles was developed by Lorio (1986).

Byers, J.A. 1995. Host tree chemistry affecting colonization in bark beetles, in R.T. Cardé and W.J. Bell (eds.). Chemical Ecology of Insects 2. Chapman and Hall, New York, pp. 154-213.