Physiological and molecular responses of lodgepole and jack pine trees under environmental stress to the blue stain fungus, Grosmannia clavigera Adriana ARANGO-‐VELEZ, Jill Hamilton1, Miranda Meents1, Dominik Royko1, L. Irina Zaharia2, Janice E.K. Cooke1 1University of Alberta, Department of Biological Sciences, Edmonton AB Canada T6G 2E9
2 NRC Plant Biotechnology InsGtute (NRC-‐PBI) / CNRC-‐InsGtut de biotechnologie des plantes (CNRC-‐IBP), Saskatoon, Saskatchewan S7N 0W9
Results & Discussion
Our objecNve was to evaluate the influence of water deficit in tree responses to G. clavigera, and idenNfy differences in consNtuNve and induced defenses between lodgepole and jack pine.
Experiments
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10
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14 dpi 35 dpi 56 dpi 68 dpi 200
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20 dpi
50 dpi
InoculaNon
Seedlings: Second year seedlings of lodgepole, and jack pine were grown in Sunshine growing media 4, and maintained under controlled condiGons as follows: 19°C (day/ night), 20-‐35% RH, and photoperiod 15h day light (200 µmol PAR) and 9h night. A total of 4 independent experiments with 8 biological replicates per experiment were sampled across treatment combinaGons. Mature trees: Natural stands of jack and planted lodgepole pines trees located in Smoky lake Alberta were used. Ten trees of similar diameter breast height (dbh) were randomly selected for each treatment combinaGon. A two by three factorial design with two treatment factors, water level and fungi inoculaNon with G. clavigera was performed.
Physiological, biochemical & transcriptional analyses: Seedling collecNon: 1, 7, 14, 28, 35, 56 and 68 days post inoculaNon (dpi) Mature tree collecNon: 20, 28, 50 and 70 dpi
A
B
10 mm
Well-‐watered Water deficit
C
Fig. 2. (A) InoculaGon and, (B) Lesion length of mature trees aeer five weeks of inoculaGon. (C) Lesion developed in pine seedlings aeer 28 days post inoculaGon.
Acknowledgements
We thank Leonard Barnhardt for providing the experimental field site at Smoky lake (AB), and also for his valuable help during the experiment. We also want to thank Dr. Walik El KLeayal and Charles Copeland for their help with this project, and Marlena Muskens for her help with the microscopy analyses.
Control
Inoculated
Control
Inoculated
Control
Inoculated
Control
Inoculated
Jack pine
70 dpi
Well watered Water deficit
Fig. 4. Decreases in photosynthesis under water deficit treatment indicates a reduced in carbon gain, therefore we hypothesize that this will influence carbon allocaNon to defense.
Lodgepole pine
Jack pine
Jack pine
PB Xylem
S PP
Xylem
C
TD
R
PP S C
10X
10X
R=rays, C=cambial zone, TD= traumaGc resin, S=sieve cells, PP=polyphenolic parenchyma cells, PB=phenolic bands.
Fig. 5. Anatomical changes of inoculated pine seedling under water deficit condiGons. InoculaGon resulted in swollen polyphenolic parenchyma (PP) cells in lodgepole pine, and development of addiGonal phenolic bands (PB) in both species, indicaNng an induced defense against G. clavigera.
Fig. 6. Hydraulic conducGvity (Kstem) as a measure of water transport, was significantly decreased in lodgepole pine, possibly due to fungal growth, fungal secondary metabolites, or tree secondary metabolites occluding tracheids. Lodgepole pine
JA (ng/g FW)
• Water deficit (WD) (<18 RWC)
Fig. 1. Experimental set up for lodgepole (A) and jack mature pines (B), and seedlings (C). Water deficit was induced by placing woven laminated polyethylene tarps.
50 dpi
TD
30
30
Lodgepole pine
3008 Genes
Jack pine
2385 Genes
2851 Genes
2574 Genes
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20
10
10 n.d.
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Control
SA (ng/g DW)
• Well watered (WW) (>40 RWC)
20 dpi
70 dpi
Fig. 3. Lesion length is generally associated with fungal growth and/or the response to the fungi. Under well-‐watered condiNons lesions were longer in lodgepole pine relaNve to jack pine, and its development is slowed under water deficit. However, aeer a longer exposure to the fungi, water deficit trees developed longer lesions, which could be a higher fungal development and a delayed tree response.
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• Wound (W) –Seedlings-‐ • Wound/G. clavigera (F) • Control (C)
Mature trees
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Water availability
14 dpi 35 dpi 56 dpi 68 dpi
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PB
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7 dpi 250
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Lodgepole pine
Seedlings
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7 dpi 250
Lodgepole pine
A
Jack pine
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A (μmol m-‐2s-‐1)
Lodgepole pine
30
Lesion length (mm)
Background Conifers exhibit both consGtuGve and induced defense mechanisms in response to aSack by pests and pathogens, such as mountain pine beetle (Dendroctonus ponderosa Hopkins) and their fungal associates. However, this defense can be modulated by environment, where abioGc stress may reduce the species ability to respond to bioGc stress. In this study, we examined the relaGve influence of water limitaGon on consGtuGve and induced defenses of lodgepole pine (Pinus contorta Dougl. ex Loud. var. la1folia) and the naïve host jack pine (P. banksiana Lamb.) to inoculaGon with the mountain pine beetle fungal associate Grosmannia clavigera. Molecular and biochemical assays evaluated inducible defense responses of jack and lodgepole pine, in response to inoculaGon with G. clavigera at 1, 7, and 28 days post-‐ inoculaGon (dpi).
0
Wound
Inoculated
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200
200
n.d.
n.d.
Control
Wound
WD+F vs WD+C
WW+F vs WW+C Inoculated Jack pine
*
4299 Genes
4014 Genes
4017 Genes
4296 Genes
100
100 0
0 Control
Wound
Inoculated
Control
Wound
Inoculated
*Significantly different at p<0.05
Fig. 7. Jasmonic acid (JA) and salicylic acid (SA) are important for defense signaling in response to insects and pathogen ahack. Fungal inoculaGon results in increased JA accumulaGon in both, jack and lodgepole pine. SA increased significantly in jack pine under fungal inoculaGon in water deficit treatment, probably as an acNve defense mechanism.
WW+F vs WW+C 1 28 1 7
7
WD+F vs WD+C 7 28 1 28 1
days post inoculaNon
7
28
Fig. 8. IdenGficaGon of differenNally expressed genes showed an early (1 and 7 dpi) inducGon of genes associated with defense-‐related processes such as terpenoid, pehenylpropanoid, and phenolic compound biosynthesis, cell wall modificaNon and hormone signaling.
Conclusions Fine-‐scale differenNaNon among funcNonal categories of differenNally expressed genes point towards species-‐specific strategies associated with defense. These data suggest that water limitaNon modulates defense response, and abioNc and bioNc factors may interact to influence the transiNon of MPB and associated pathogens from lodgepole pine to jack pine.