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METHODS
Antioxidant Alteration Of Ash Antixenosis To Malacosoma disstria
Because sulfhydryl groups may readily participate in redox reactions in living cells (Morton 1965), our studies
evaluated a natural antioxidant (reducing agent), o_-tocopherol(vitamin E), as an elicitor of defensive phytochemistry. Alphatocopherol
was tested in a basal trunkband on green ash trees to alter their antixenosis to forest tent caterpillar larvae,
Malacosoma disstria (Hubner). The trees were 4.6m tall 'Summit' green ash, Fraxinus pennsylvanica var. subintegerrinia
(Vahl) Fernald, and had a mean trunk diameter of 5.1 cm at 1 m above the soil surface. The basal trunk of each tree received
o_-tocophero[, a proven elicitor (Neupaneand Norris 1991b), as a band application at the dosages of 25.0 or 50.0 IU / ml in
mineral oil. An international unit (IU) equals 1 mg of all-rac-cx-tocopheryl acetate (U.S. Pharmacopaea 1980). Sixty ml of
either concentration of ot-tocopherol in mineral oil were placed on a 120cm 2bandage; the control trees received only 60 ml of
mineral oil.
Insect Bioassays
Leaves for bioassay were removed from three distinct trees per treatment or control at each of several intervals after
treatment, and two-choice feeding assays were conducted with 1.5-cm-diam disks cut with a No. 8 cork borer from such
leaves and with third-instar forest tent caterpillars. The insect's feeding option thus was between a comparable leaf disk from
an elicited versus a solvent-control tree. The quantitation of insect feeding was detailed by Markovic et al. (1993).
Chemical Analyses
[.,eaves for chemical analyses were collected from three distinct ash trees for each elicitation dosage and the solvent
control, and for 8 and 16days after treatment. Three compound leaves from each tree were immediately put individually into
a glass jar containing 80% methanol, and then stored in darkness at -20°C until chemical analysis. Procedures used to extract
chemicals from ash leaves; hydrolyze extracted chemicals; and analyze the chemicals by high performance thin layer
chromatography (HPTLC) and high performance liquid chromatography (HPLC) were as detailed by Markovic et al. (1993).
RESULTS
Antioxidant Altered Foliar Antixenosis In Ash
Forest tent caterpillar herbivory was altered on leaf disks from green ash trees elicited with either of the two dosages
of c_-tocopherol as compared to disks from solvent-treated ash trees (Table 1). High performance thin layer chromatography
(HPTLC) revealed distinct differences in both the non-hydrolyzed and hydrolyzed portions of the ethyl acetate extractables
from elicited versus control ash trees (Markovic et al. 1993). Reduced insect preference for foliage due to tocopherol
elicitation was accompanied by an increased mean total HPLC-resolved peak area of ethyl acetate extractables from the nonhydrolyzed
leaf sample as compared to control foliage. Whereas, increased insect preference for foliage due to elicitation
was accompanied by a decreased total HPLC-resolved peak area of such extractables as compared to control. HPLC of the
above non-hydrolyzed, ethyl acetate extractables showed mainly quantitative, rather than qualitative, differences between ¢xtocopherol-elicited
versus solvent-treated controls. The differences were especially evident in five major, and three lesser,
peaks; these eight peaks thus were chosen for a more detailed comparison between the non-hydrolyzed fraction of the ethyl
acetate extractables from the two cx-tocopherol treatments and solvent-treated controls at two times (8 and 16 days) after
elicitation.
At 8 days after elicitation, the foliage from trees receiving 25 IU / ml contained significantly more of HPLC peaks
1-6, but not of peaks 7 or 8, than did leaves from solvent-control trees (Fig. 3). Foliage from these treated trees also was
significantly less preferred than that from the control trees (Table 1). Conversely, at 16 days after elicitation, foliage from
trees that received either 25 or 50 IU / ml was preferred over that from the solvent-control ones (Table 1). The ethyl acetate
extractables at 16 days after elicitation from preferred foliage from trees that received 25 or 50 IU / ml had a smaller average
total HPLC-resolved peak area than did those from the leaves of solvent-control trees (Fig. 4). The mean total HPLC-
resolved peak area was also significantly different between the foliage collected from the solvent-control trees at 8 and 16
days after elicitation. Thus, time in thegrowing season also affected the chemical composition of the trees.
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