Table of contents
1. Abstract
2. Introduction
3. Objective of the Study
4. Effects of Phenolic compounds and Monoterpenes on Spore Germination
5. Effects of Phenolic compounds and Monoterpenes on Mycelial
Growth
6. Discussion
7. Conclusion
8. References
Abstract
Red pine decline, characterized by an expanding circular area of dead
and declining trees, is becoming
increasingly prevalent in Lake States plantations. The root collar
weevil-Hylobius radicis, the pales weevil-
Hylobius pales, the pitch-eating weevil-Pachylobius picivorus,
the red turpentine beetle-Dendroctonus
valens and Hylastes porculus were significantly more
abundant in declining stands than in healthy Pinus
resinosa stands. These root- and lower stem-infesting insects consistently
carried Leptographium
terebrantis and Leptographium procerum. Higher soil organic
matter, pH and K levels were also associated
with areas of mortality. The in vitro effects of a red pine phenolic
compound (pinosylvin), a phenolic
compound common to other species (tannic acid), and the major red pine
monoterpenes (alpha-pinene, beta-
pinene, and delta-carene) on spore germination and mycelial growth
of Sphaeropsis sapinea were examined.
Two A and two B morphotype isolates were used. At 88 micro gram/mm2,
pinosylvin inhibited spore
germination of all four isolates (98 to 100%). At 8.8 micro gram/ mm2,
spore germination of B isolates was
inhibited more than that of A isolates (73 versus 30%). Pinosylvin
also inhibited mycelial growth of B
isolates more than that of A isolates. Tannic acid stimulated
or had little affect on spore germination and
had little affect on mycelial growth of either morphotype. Spore germination
of B isolates was inhibited
more than that of A isolates by beta-pinene at saturation (79 versus
37%). Spore germination of B isolates was
inhibited and germination of A isolates was stimulated by delta-3-carene
below saturation (49 versus -7%).
Mycelial growth of B isolates was inhibited more than that of
A isolates by all monoterpenes at saturation.
Differences observed between morphotypes below saturation were significant
only for beta-pinene. These
results demonstrate the biological activity of a phenolic compound
and monoterpenes that occur in red
pine. The differential responses might provide means of distinguishing
morphotypes and offer a potential
explanation for ecological specialization.
Introduction
In several coniferous tree species, monoterpenes( Russell and Berryman,1976;
Cates and
Alexander,1982; Raffa and Berryman,1982,1983; Cook and Hain,1986; Paine
et al.1987;
Lewinsohn et al.1991a) and phenolics( Jorgensen,1961; Shain,1967; Lieutier,1993)
occur
in increased concentrations in host tissue following failed attack
by bark beetles and their
associated fungi. Hence, these compounds have been hypothesized to
exert defensive
pressures against both insects and pathogens. Supporting evidence includes
inhibition of
mycelial growth of beetle-vectored fungi by monoterpenes as well as
toxic and/or
repellent effects on bark beetles( Smith,1963; Cobb et al.1968; Shrimpton
and
Whitney,1968; DeGroot,1972; Coyne and Lott,1976; Bordasch and Berryman,1977;
Raffa et al.1985; Bridges,1987; Delmore and Lieutier,1990; Raffa and
Smalley,1995).
Recently different studies demonstrated inhibition of fungal propagule
germination,
mycelial growth, and beetle tunneling by monoterpenes and inhibition
of mycelial growth
by phenolics that are found in red pine resin.
Critical gaps remain in our knowledge of the dynamics of conifer chemical
defenses.
Although trees can usually resist attack by most parasitic organisms,
numerous examples
exist of widespread mortality caused by beetle-fungal complexes. In
many of these cases,
mortality appears to follow some biotic and abiotic stress( Nebeker
et al.1993). Such
relationships may provide insights into basic theories of plant defense
and of insect-
microbial complexes that jointly encounter host resistance.
Objective of Study
Sphaeropsis sapinea causes shoot blight and canker diseases of pines
and other conifers
throughout the world. Two S. sapinea morphotypes (A and B) occur in
the north central
United States( Palmer et al.1987). Morphotypes are groups of
individuals of a species
with unknown or no taxonomic significance that can be morphologically
differentiated(
Hawksworth et al.1995). On red pine (Pinus resinosa), isolates of the
A morphotype are
more aggressive than isolates of the B morphotype( Blodgett et al.1997).
Phenolic compounds and monoterpenes are hypothesized to be the main
categories of
chemicals involved in the resistance of pines to several fungal pathogens
and insects(
Klepzig et al.1995; Lieutier et al.1988; Raffa et al.1991; Shrimpton
et al.1973). These
chemicals can occur in high concentrations in conifer species(up to
several hundred
milligrams per gram dry weight). They also can increase in abundance
when trees are
damaged mechanically or colonized by a pathogen. Monoterpenes are associated
with the
resistance of P.radiata to S.sapinea( Chou et al.1976) and the resistance
of P.elliottii to
Cronartoum quercuum f. sp. fusiforme has been correlated with the concentration
of ?-
phellandrene in bark( Rockwood, D.L.1974). However, Franich et al.1982
found little
relationship between the monoterpene composition of P. radiata and
resistance of mature
trees to Dothistroma septospora.
The fungistatic properties of phenolics and monoterpenes have been documented
in vitro.
Phenolics and monoterpenes are inhibitory at concentrations observed
in pines and other
conifer species( Alcubilla et al.1971; Hergert, H.L.1960; Klepzig et
al.1996; Raffa et al.
1991). Previous studies have shown that monoterpenes inhibit a number
of pine
pathogens, including Heterobasidion annosum,( Cobb et al.1968; Gibbs,
J.N.1968;
Kennedy, R.W.1956) Leptographium spp.( Klepzig et al.1996), Ophiostoma
spp.( Raffa,
K.F.1991), D.septospora( Franich et al.1982) and Ceratocystis
spp.( Cobb et al.1968).
Tolerances to monoterpenes differ among species of decay and stain
fungi( DeGroot,
R.C.1972; Hintikka, V.1970). Monoterpenes also inhibit a S. sapinea
isolate of unknown
morphotype( Chou et al.1976).
Both phenolics and monoterpenes were studied in vitro. Phenolics included
pinosylvin,
the most common phenolic of red pine( Jorgensen, E.1961) and tannic
acid, a common
phenolic in other species. Monoterpenes included the three most common
in red pine: ?-
pinene, ?-pinene, and ?-3-carene. These comprise 59 to 74, 13 to 33,
and 1 to 5%, respectively,
of the total monoterpenes in these species( Blodgett, J.T.1996). The
objectives were to
examine (I) the effects of these phenolics and monoterpenes on spore
germination and mycelial
growth of S. sapinea and (ii) potential differences in the responses
of the two
morphotypes to these chemicals. The null hypothesis tested in this
study were that (I)
pinosylvin and monoterpenes have no affect on germination and growth
of S. sapinea and
(ii) morphotypes do not differ in their tolerance to these chemicals.
Effects of Phenolic compounds and Monoterpenes on Spore Germination
Blodgett et al. (1997) examined the effects of phenolic compounds and
monoterpenes
on spore germination. Conidia were produced for two monoconidial
isolates of each
morphotype. Debarked and autoclaved red pine twig sections were placed
on 1.5%
water agar plates that were inoculated with colonized 1.5% WA plugs.
Plates were
incubated for 5 weeks at ambient laboratory temperature (?24?C) and
light. Twigs on
which pycnidia developed were scraped with a scalpel and washed with
sterile distilled
water to release conidia. The resulting suspension were filtered through
two layers of
sterile cheesecloth. Conidial concentrations were estimated with a
hemacytometer and
adjusted to ?1.25x103 condida per ml with sterile distilled water.
Conidial suspensions
were kept on ice to prevent spore germination between collection, quantification,
and plating.
Pinosylvin was obtained from Forest Products Laboratory. Commercially
avilable tannic
acid was used. Phenolics were dissolved in 80% aqueous acetone (vol/vol)
at
concentration of 0.2 and 0.02 mg/ml. The solutions were applied and
allowed to dry on
the surface of 2% WA in multiwell tissue-culture plates at 88 and 8.8
?g/mm2 . Eighty
percent acetone was applied for controls. The acetone was allowed to
evaporate in a
laminar-flow hood. Conidial suspensions (80 ?l per well) of each isolate
were added.
Commercially obtained monoterpenes included (+/-)-?-pinene (98%), 1-(S)-(-)-?-
pinene(99%), and ?-3-carnene(95%). Saturated atmospheres and vapor
concentrations
below saturation (70 and 10% of saturation) were compared. Different
vapor
concentrations were used, because the nonpolar, volatile monoterpenes
cannot be
dissolved in polar solvents. The conidial suspensions were added at
80 ?l per well to the
multiwell tissue-culture plates containing 2% WA. Monoterpenes were
placed on filter
paper attached with paraffin wax to the lid above each well, and plates
were quickly
sealed. A single monoterpene at a single concentration was used for
each plate. No
monoterpenes were placed in control plates.
Vapor concentrations were estimated by gas-liquid chromatography (GLC)
by applying
various amounts of the monoterpenes to filter paper attached to the
lid of the tissue-
culture plates containing 2% WA. GLC was performed using a modification
of the
procedures with a Schimadzu GC-9A gas chromatograph with flame ionization
detector.
Oven temperature was set at 60?C, injector and detector temperature
was 220?C, and the
carrier gas helium at 30 cm/s, with a methane retention time of 84
s. Vapor
concentrations below saturation were estimated by peak integration
with the Schimadzu
C-R3A digital integrator and relative responses to the saturated atmospheres.
Both the phenolic and monoterpene plates were wrapped individually
with plastic wrap
and sealed inside two plastic bags. Plates were incubated at 25?C in
the dark for 10 h,
after which germination was stopped by adding 0.1 ?g of thimersol per
ml. Plates were
stored at 4?C in the dark between germination counts. Percent germination
was
determined from a minimum count of 50 spores per well (range 50 to
89). Four replicates
for each combination of isolate, chemical, and concentration were used.
Pinosylvin inhibited germination of both morphotypes, but tannic acid
had little effect on
germination (Fig.1). The average percent germination for controls was
56 ? 2.3%
standard error (SE) for A isolates and 45 ? 2.6% SE for B isolates.
At the highest
concentration, pinosylvin inhibited germination of all isolates with
no differences
between morphotypes. Germination was less inhibited at one-tenth concentration,
but B
isolates were inhibited more than A isolates ( P= 0.002). Tannic acid
either stimulated
germination or had little influence on either morphotype at either
concentration.
Saturated atmospheres of the monoterpenes inhibited germination of
both morphotypes.
The average percent germination for controls was 62 ? 1.9% SE for A
isolates and 49 ?
2.4% SE for B isolates. Responses of morphotypes differed only at saturated
atmosphere
for ?-pinene ( p = 0.001) and at 70% saturation for ?-3-carene ( p<0.001).
In both cases,
B isolates were inhibited more than A isolates. There was little inhibition
and
morphotypes could not be differentiated at 10% of saturation. Compared
to other
monoterpenes, ?-pinene was more inhibitory at saturation( Blodgett
et al.1997).
Effects of Phenolic compounds and Monoterpenes on Mycelial Growth
Blodgett et al. (1997) also examined the effects of phenolic compounds
and monoterpenes
on mycelial growth. The phenolics (pinosylvin and tannic acid)
were dissolved in 80% aqueous acetone
(vol/vol) at concentrations of 0.2, 0.02, and 0.002 mg/ml. The solutions
were applied and
allowed to dry on the surface of 2% WA in petri plates at 88, 8.8,
and 0.88 ?g/mm2 .
Eighty percent acetone was applied for controls. The acetone was allowed
to evaporate in
a laminar-flow hood. Colonized 1.5% WA plugs, 4 mm in diameter, were
cut from
margins of actively growing cultures and placed fungus side down at
the edge of the agar
in the petri plates. Plates were wrapped with paraffin. The isolates
used were the same
ones described above.
Monoterpenes were placed on filter paper at the bottom of 2% WA slants
that had rubber-
lined screw-top closures. Monoterpene vapor concentrations were estimated
by GLC with
commercially obtained monoterpenes. Saturated atmospheres and vapor
concentrations
below saturation ( 80, 66, and 32% of saturation) were compared.
Colonized 1.5% WA
plugs, 4 mm in diameter, were cut from margins of actively growing
cultures and placed
fungus side down on agar at the top of the slants. The slants were
sealed, creating
saturated atmospheres. No monoterpenes were placed on filter paper
in control slants.
Plates and slants were incubated at 25?C in the dark. Radial growth
of the cultures was
recorded at two weeks. Five replicates for each combination of isolate,
chemical, and
concentration were used.
Pinosylvin inhibited radial growth of both morphotypes, and tannic acid
had little effect
on growth of either morphotype. The average mycelial growth for controls
was 46 ? 0.9
mm SE for A isolates and 42 ? 0.5 mm SE for B isolates. Pinosylvin
inhibited mycelial
growth of B isolates more than A isolates at both 0.2 and 0.02 mg/ml
( p< 0.001). There
was little inhibition and morphotypes could not be differentiated at
0.002 mg/ml. Tannic
acid had a similar influence on A isolates and was less inhibitory
to B isolates compared
to pinosylvin at all concentrations.
The monoterpenes inhibited radial growth of both morphotypes. However,
all three
monoterpenes inhibited radial growth of B isolates more than
A isolates at saturated
atmospheres (p< 0.001). The average mycelial growth for controls
was 35 ? 1.2 mm SE
for A isolates and 44 ? 1.1 mm SE for B isolates. Differences between
morphotypes at
both 80 and 66% of saturation were significant only for ?-pinene (p<0.001).
There was
little inhibition at 66 and 33% of saturation for any of the monoterpenes(
Blodgett et
al.1997).
Discussion
Examination of the effects of host phenolic compounds and monoterpene
may provide
additional means of differentiating morphotypes of S. sapinea.
The two S. sapinea
morphotypes have different colony morphology, growth rates on potato
dextrose agar,
mean spore sizes, and isozymes( Palmer et al.1987). S. sapinea isolates
also can be
differentiated into morphotypes using random amplified polymorphic
DNA analysis(
Smith et al.1995). The differential inhibition of these two morphotypes
by monoterpenes
and pinosylvin provides further justification for distinguishing different
types and might
help explain differences in the response of red pine to A and B morphotypes.
Previously reported differences in aggressiveness between morphotypes
on red pine(
Blodgett J.T.1996) may result from variable responses to host chemicals.
The bioassays
show that pinosylvin and several monoterpenes that occur in red pine
inhibit A and B
morphotypes of S. sapinea in vitro. Although these chemicals
are inhibitory to S.
sapinea, the isolates tested differ in their tolerances to these chemicals.
The inhibitory effects of ?- and ?-pinene on A isolates were similar
to those for an isolate
of unknown morphotype( Chou et al.1976). Monoterpenes were more inhibitory
to germ
tube growth than to germination for a single S. sapinea isolate of
unknown morphotype.
In the current study, mycelial growth was inhibited more than spore
germination when S.
sapinea was exposed to the monoterpenes. In the previous study, germ
tube growth over
6h was reduced by ?- and ?-pinene and ?-3-carene by ?70 to 80% at saturated
atmospheres( Chou et al.1976) , which is similar to percentages observed
for mycelial
growth of A isolates in this study. The ?-3-carene is found to be the
most inhibitory of the
monoterpenes on spore germination.. In this study, there were no clear
differences
between monoterpenes.
Phenolic extracts of red pine are inhibitory to other fungal pathogens.
Consistent with the
results of B isolates, Klepzig et al.1996 found that phenolic extracts
of red pine (primarily
pinosylvin and its monomethyl ether) inhibited mycelial growth of L.
terebrantis and L.
procerum. In this study, mycelial growth of B isolates was substantially
inhibited by
pinosylvin, but there was little effect on A isolates. Although in
this study germination of
both morphotypes was substantially reduced by pinosylvin, phenolic
extracts of red pine
did not affect germination of the Leptographium spp.( Klepzig et al.1996).
Thus, the
effects of red pine phenolics may vary with different pathogens or
assay conditions.
Differences between morphotypes in response to these chemicals are
consistent with
differences in aggressiveness on red pine and might offer evidence
for ecological
specialization of the morphotypes. Conifers react to colonization by
pathogens by
forming resinous lesions in phloem and sapwood around infection sites(
Klepzig et
al.1995; Nebeker et al.1993; Raffa, K.F.1991). The B types are restricted
in red pine to
the immediate vicinity of the inoculation site, but A isolates can
spread, girdle, and kill
red pine shoots( Blodgett et al.1997). Pinosylvin and monoterpenes
may inhibit the
growth of B isolates, preventing colonization. Collection of isolates
of the B morphotype
only from wounded trees suggest they are opportunistic colonizers of
damaged or
weakened host tissues( Palmer et al.1987; Wingfield et al.1983). The
success of the B
morphotype in these tissues may reflect inhibition of normal pine defensive
responses,
including reductions in normally inhibitory concentrations of red pine
monoterpenes and
phenolics.
Conclusion
It is hypothesized that monoterpenes and phenolics play a role in the
defensive response
of red pine against insect-fungal attack, that stress may predispose
red pine to attack by
insect-fungal complexes, and that such interactions are involved in
red pine decline
disease.
References
1. Alcubilla, M., Diaz-Palacio, M.P., Kreutzer, K., Laatsch, W., Rehfuess,
K.E., and
Wenzel, G. 1971. Beziehungen zwischen..... European Journal of Forest
Pathology.1:
100-114.
2. Blodgett, J.T. 1996. The effects of host water stress on disease
development by
different Sphaeropsis sapinea morphotypes. Ph.D. dissertation. University
of
Wisconsin-Madison, Madison
3. Blodgett-JT, Stanosz-GR. 1997. Sphaeropsis sapinea morphotypes differ
in
aggressiveness, but both infect nonwounded red or jack pines. Plant
Disease. 81: 143-
147.
4. Blodgett-JT, Stanosz-GR. 1997. Differential Inhibition of Sphaeropsis
sapinea
Morphotypes by a Phenolic Compound and Several Monoterpenes of Red
Pine.
Phytopathology. 87 p.606-609.
5. Bordasch, R.P., and Berryman, A.A. 1977. Host resistance to the fir
engraver beetle.
Canadian Entomology. 109: 95-100.
6. Bridges, J.R. 1987. Effects of terpenoid compounds on growth of symbiotic
fungi
associated with the southern pine beetle. Phytopathology. 77: 83-85.
7. Cates, R.G., and Alexander, H. 1982. Host resistance and susceptibility,
pp. 212-263.
A.P.S. Press, St. Paul, Minnesota.
8. Chou,C.K.S., and Zabkiewicz, J.A. 1976. Toxicity of monoterpenes
from Pinus
radiata cortical oleoresin to Diplodia pinea spores. Eur. J. For. Pathol.
6: 354-359.
9. Cobb, F.W., Jr., Krstic, M., Zavarin,E., and Barber, H.W.,Jr. 1968.
Inhibitory effects
of volatile oleoresin components on Fomes annosus and four Ceratocyctis
species.
Phytopathology 58: 1327-1335.
10. Cook, S.P., and Hain, F.P. 1986. Defensive mechanisms of loblolly
and short leaf
pine against attack by southern pine beetle and its fungal associate.
Journal of
Chemical Ecology. 12: 1397-1406.
11. Coyne, J.F., and Lott, L.H. 1976. Toxicity of substances in pine
oleoeresin to
southern pine beetles. Journal Ga. Entomol. Soc. 11: 301-305.
12. De Groot, R.C. 1972. Growth of wood-inhabiting fungi in saturated
atmospheres of
monoterpenoids. Mycologia 64: 863-870.
13. Delorme, L. and Lieutier, F. 1990. Monoterpene composition of the
preformed and
induced resins of Scots pine, and their effect on bark beetles and
associated fungi.
European Journal of Forest Pathology. 20: 304-316.
14. Franich,R.A.,Gaskin,R.E., WellsL.G., and Zabkiewicz, J.A. 1982.
Effects of Pinus
radiata needle monoterpenes on spore germination and mycelial growth
of
Dothistroma pini in vitro in relation to mature tree resistance. Physiol.
Plant Pathol.
21: 55-63.
15. Gibbs, J.N. 1968. Resin and the resistence of conifers to Fomes
annosus. Ann.Bot.
32: 649-665.
16. Hawksworth,D.L., Kirk,P.M., Sutton, B.C., and Pegler,D.N.
1995. Ainsworth and
Bisby's Dictionary of the Fungi. 8th ed. International Mycological
Institute, Egham,
England.
17. Hergert,H.L. 1960. Chemical composition of tannins and polyphenols
from conifer
wood and bark. For.Prod.J. 10: 610-617.
18. Hintikka,V. 1970. Selective effect of terpenes on wood-decomposing
Hymenomycetes. Karstenia 11: 28-32.
19. Jorgensen,E. 1961. The formation of pinosylvin and its monomethyl
ether in the
sapwood of Pinus resinosa Ait. Can. J. Bot. 39: 1765-1772.
20. Kennedy, R.W. 1956. Fungicidal toxicity of certain extraneous compounds
of
Douglas-fir heartwood. For. Prod. J. 6: 80-84.
21. Klepzig,K.D., Smalley, E.B., Raffa, K.F. 1996. Combined chemical
defenses against
an insect-fungal complex. Journal of Chemical Ecology. 22: 1367-1388.
22. Klepzig,K.D., Kruger, E.L., Smalley, E.B.,and Raffa, K.F. 1995b.
Effects of biotic
and abiotic stress on induced accumulation of terpenes and phenolics
in red pines
inoculated with bark beetle vectored fungus. Journal of Chemical Ecology.
21 p. 601-
626.
23. Lewinsohn, E., Guzen, M., Savage, T.J., and Croteau, R. 1991a. Defense
mechanisms
of conifers: Relationships of monoterpene cyclase activity to anatomical
specialization and oleoresin monoterpene content. Plant Physiology.
96: 38-43.
24. Lieutier, F. 1993. Induced defense reaction of conifers to bark
beetles and their
associated Ophiostoma species, pp.225-233. A.P.S. Press. St. Paul,
Minnesota .
25. Lieutier, F., and Berryman, A.A. 1988. Preliminary histological
investigations of the
defense reactions of three pines and two chemical elicitors. Canadian
Journal of
Forest Resources. 18: 1243-1247.
26. Nebeker, T.E., Hodges, J.D., and Blanche, C.A. 1993. Host response
to bark beetle
and pathogen colonization. pp.157-173. in: Beetle-Pathogen Interactions
in conifer
forests. Academic Press, New York.
27. Paine, T.D., and Stephen, F.M. 1987. Influence of tree stresses
and site quality on the
induced defensive system of loblolly pine. Canadian Journal of Forest
Resources. 17:
569-571.
28. Palmer. M.A.., Stewart,E.L., and Wingfield, M.J. 1987. Variation
among isolates of
Sphaeropsis sapinea in the north central United States.
Phytopathology 77: 944-948.
29. Raffa,K.F. 1991. Induced defensive reactions in conifer-bark beetle
systems. Pages
245-276 in: Phytochemical Induction by Herbivores. D. W. Tallamy, ed.
John Wiley
& Sons, New York.
30. Raffa, K.F., and Berryman, A.A. 1982. Accumulation of monoterpenes
and associated
volatiles following fungal inoculation of grand fir with a fungus vectored
by the fir
engraver. Canadian Entomology. 114: 797-810.
31. Raffa, K.F., and Berryman, A.A. 1983. Physiological aspects of lodgepole
pine
wound responses to a fungal symbiont of the mountain pine beetle. Canadian
Entomology. 115: 723-734.
32. Raffa, K.F., and Smalley, E.B. 1995. Interaction of pre-attack
and induced
monoterpenes in conifer defense against bark beetle-fungal complexes.
Oecologia. In
press.
33. Raffa, K.F., Berryman, A.A., Simasko, J., Teal, W., and Wong,
B.L. 1985. Effects
of grand fir monoterpenes on the fir engraver and its symbiotic fungus.
Environmental Entomology. 14: 552-556.
34. Rockwood., D.L. 1974. Cortical monoterpene and fusiform rust resistance
relationships in slash pine. Phytopathology. 64: 976-979.
35. Russell, C.E., and Berryman, A.A. 1976. Host resistance to the fir
engraver beetle. 1.
Monoterpene composition of pitch blisters and fungus-infected wounds.
Canadian
Journal of Botany. 54: 14-18.
36. Shain, L. 1967. Resistance of sapwood in stems of loblolly pine
to infection by
Fomes annosus. Phytopathology. 57: 1034-1045.
37. Shrimpton, D.M., and Whitney, H.S. 1968. Inhibition of growth of
blue stain fungi by
wood extractives. Canadian Journal of Botany. 46: 757-761.
38. Shrimpton, D.M. 1973. Extractives associated with wound response
of lodgepole pine
attack by the mountain pine beetle and associated microorganisms. Canadian
Journal
of Botany. 51: 527-534.
39. Smith, R.H. 1963. Toxicity of pine resin vapors to three species
of Dendroctonus bark
beetles. Journal Econ. Entomol. 56: 827-831.
40. Smith,D.R., and Stanosz, G.R. 1995. Confirmation of two distinct
populations of
Sphaeropsis sapinea in the north central United States using RAPDs.
Phytopathology
85: 699-704.
41. Wingfield, M.J., and Plamer, M.A. 1983. Diplodia pinea associated
with insect
damage on pines in Minnesota and Wisconsin. pp. 249 in: Proc. 4th Int.
Congr. Plant
Pathology. University of Melbourne, Australia.
2
18