Combined Chemical Defense of Red Pines Against Insect-Fungal Attack

Table of contents


1. Abstract						P. 3
2. Introduction						P. 4
3. Objective of the Study				P. 4-6
4. Materials and Methods
	(i) Effects on Spore Germination		P. 6-8
	(ii) Effects on Mycelial Growth			P. 8-9
	(iii) Statistical Analysis			P. 9
5. Results
	(i) Effects on Spore Germination		P. 9-10
	(ii) Effects on Mycelial Growth			P. 10-11
6. Discussion						P. 11-13
7. Conclusion						P. 13
8. References						P. 14-15
			

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 ([DHSG1](-pinene, (-pinene, and (-
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 (-pinene at saturation (79 versus 37%). Spore germination of B 
isolates was inhibited and germination of A isolates was stimulated by (-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 (-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 and phenolics 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. 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. 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. Morphotypes are groups of  individuals of a species 
with unknown or no taxonomic significance that can be morphologically 
differentiated. On red pine (Pinus 
resinosa), isolates of the A morphotype are more aggressive than isolates of the 
B morphotype.
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. 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, and 
the resistance of P.elliottii to Cronartoum quercuum f. sp. fusiforme has been 
correlated with the concentration of (-phellandrene in bark. However, Franich 
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. Previous studies have shown that monoterpenes 
inhibit a number of pine pathogens, including Heterobasidion annosum, 
Leptographium spp., Ophiostoma spp., D.septospora, and Ceratocystis spp. 
Tolerances to monoterpenes differ among species of decay and stain fungi. 
Monoterpenes also inhibit a S. sapinea isolate of unknown morphotype.
Both phenolics and monoterpenes were studied in vitro. Phenolics included 
pinosylvin, the most common phenolic of red pine 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. 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.


Materials and Methods

(i) Effects 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.

(ii) Effects 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.


(iii) Statistical Analysis :
For comparisons of germination and mycelial growth, inhibition percentages were 
calculated (% inhibition =(control-treatment)/control x 100). Inhibition 
percentages were analyzed by one-way analysis of variance (ANOVAs). If 
significant differences were found ( P ( 0.05), means were separated by Fisher's 
least significant difference (LSD) at P= 0.05. ANOVAs (using the general linear 
model procedure) were performed with the Minitab for windows program.

Results

 (i) Effects on Spore Germination :
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.

(ii) Effects on Mycelial Growth :
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.

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. S. sapinea isolates also can be 
differentiated into morphotypes using random amplified polymorphic DNA analysis. 
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 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. 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, 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 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. 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. 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. 
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. 
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.


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