Final Report Summary - SYBHES (Systems biology of the Heterobasidion-spruce interaction: application of metabolomics and genomics to understanding host resistance)
In economic terms, root and butt rot of conifers caused by Heterobasidion is the most destructive endemic disease in European forestry. Despite having an impact estimated at an excess of EUR 1 billion per annum, control measures available in forests with a long history of management are limited. In previous European Union (EU)-supported work (RESROBS; QLRT- 2000-00241), we and others demonstrated that individual spruce trees show marked variations in susceptibility to Heterobasidion (Bodles et al., 2006, 2007; Woodward et al., 2007). Resistance to pathogen growth was genetically inherited, and with better understanding, it could be exploited in spruce breeding programmes.
In SYBHES, we utilised state-of-the-art metabolomic and genomic methods to examine precisely how Sitka spruce clones differ in terms of chemistry and gene regulation when infected with Heterobasidion.
(1) Metabolite fingerprinting of Picea sitchensis bark and sapwood was carried out using proton (1H) nuclear magnetic resonance (NMR) after wounding and artificial inoculation with the white rot fungus Heterobasidion annosum sensu stricto. The aim was to determine whether metabolites would differ in clones showing differing levels of susceptibility to the pathogen, in the fungal compared to the control treatment (wounding, no fungus) and the reference (healthy sample collected at the time of wounding and inoculation), at two different host locations, and at different sampling times (3 and 43 days).
The results of this work suggested that different metabolic processes occur in bark and sapwood after wounding and fungal inoculation, compared to healthy samples collected before treatment: in bark, greater peaks were elicited in the aromatic region, whereas in sapwood lower concentrations of all metabolites were observed in inoculated samples, compared to healthy samples. Multivariate statistical analysis carried out with principal component analysis showed highly significant effects of reference, location, and time, and significant effects of clone and fungus. Differences between clones were apparent in sapwood but not in bark, and were due to peaks in the aliphatic region. Over time, in bark there was a decrease in carbohydrate peaks, followed by an increase in aliphatic and aromatic peaks. Sapwood, by contrast, showed a decrease in all peaks, followed by an increase in carbohydrate and aliphatic peaks. Changes in carbohydrate levels were observed within the lesion compared to more distal location in both bark and sapwood.
(2) Expression profiles of a range of defence-related genes, including phenylalanine ammonia lyase (PAL), cinnamoyl-CoA reductase (CCR1), hydroxycinnamoyl CoA shikimate / quinate hydroxycinnamoyltransferase (HCT1) and cinnamyl alcohol dehydrogenase (CAD) of the phenylpropanoid pathway, peroxidase (PaPX3), and a class IV chitinase (PaCHI4), with a-tubulin used as endogenous control, were investigated in bark and sapwood of Picea sitchensis clones showing similar susceptibility to Heterobasidion annosum s.s. 3 days after wounding and artificial inoculation with the pathogen. Constitutive levels of expression were compared with levels induced after treatment at two locations (at the site of inoculation and distally, 1 cm away from the inoculation site). In addition, lignin and soluble and cell wall bound phenolic compounds were extracted from the distal zone only, after inoculation.
Compared to constitutive levels, metabolic processes differed between bark and sapwood: after treatment, in bark all genes were up-regulated, whereas in sapwood all genes except for PaPX3 and PaCHI4 were downregulated. In bark all genes investigated, except for CAD, were expressed at greater levels at the site of inoculation than in the distal zone, whereas in sapwood PAL, CCR1, HCT1 and CAD were present at lower levels around the site of inoculation than in the distal zone. Furthermore, compared to wounding only, inoculation with H. annosum triggered significantly different CAD, PaPX3, and PaCHI4 levels in bark but not in sapwood, indicating differences in the defence response between these two tissues. Different quantities of lignin and soluble and cell wall bound phenolic compounds were found in bark and sapwood. In bark, wounding and inoculation led to different concentrations of cell wall bound phenolic compounds (unknown2, unknown3, coniferin, astringin, taxifolin, piceid, and isorhapontin) compared to healthy control samples, whereas in sapwood, concentrations did not differ following treatment.
These results indicated that bark of Sitka spruce has a stronger and earlier response to wounding and pathogen inoculation than sapwood.
Potential impact
These results are of high significance in the forest industry throughout the northern hemisphere. The work has demonstrated the different techniques can be utilised to detect levels of susceptibility / resistance to Heterobasidion annosum. Compared with long-term susceptibility trials, these techniques provide rapid methods for susceptibility testing in newly available spruce clones so that plants showing greater resistance to the pathogen can be used on forest sites conducive to disease development. Further testing is required to fully corroborate the methods developed in this work against those used in younger plants in the earlier work (Bodles et al., 2006, 2007; Woodward et al., 2007). It is also important to note that, following confirmatory testing, the results obtained in this work will be equally applicable to other important species of conifer in the Pinaceae. Securing forest productivity in this manner has extensive socio-economic impacts in terms of retaining rewarding employment in the rural sector, improving yields from European forests and increasing quality timber output, thereby improving competitiveness of European forestry industries in the world markets.
Contacts:
Dr Giuliana Deflorio and Dr Steve Woodward
Institute of Biological and Environmental Sciences
University of Aberdeen
Scotland, United Kingdom
Professor Marcel Jaspars
Department of Chemistry
University of Aberdeen
Scotland, United Kingdom
Dr Carl Gunnar Fossdal
Norwegian Institute of Forest and Landscape
Ås, Norway
email: s.woodward@abdn.ac.uk
Tel.: +44-122-4272669
References:
- Bodles, W. J. A., Fossdal, C. G. and Woodward, S. (2006). Multiplex real-time PCR detection of pathogen colonisation in the bark and wood of Picea sitchensis clones with different levels of resistance to Heterobasidion annosum. Tree Physiology 26: 775-782.
- Bodles, W. J. A., Beckett, E. and Woodward, S. (2007) Responses of Sitka spruce from different origins to inoculation with Heterobasidion annosum: Fungal growth, lesion development resin duct development and lignosuberised boundary zone formation. Forest Pathology 37:174-186.
- Woodward, S., Bianchi, S., Bodles, W. J. A., Beckett, E. and Michelozzi, M. (2007). Physical and chemical responses of Sitka spruce (Picea sitchensis) clones to colonisation by Heterobasidion annosum as potential markers for relative host susceptibility. Tree Physiology 27:1701-1710.
In SYBHES, we utilised state-of-the-art metabolomic and genomic methods to examine precisely how Sitka spruce clones differ in terms of chemistry and gene regulation when infected with Heterobasidion.
(1) Metabolite fingerprinting of Picea sitchensis bark and sapwood was carried out using proton (1H) nuclear magnetic resonance (NMR) after wounding and artificial inoculation with the white rot fungus Heterobasidion annosum sensu stricto. The aim was to determine whether metabolites would differ in clones showing differing levels of susceptibility to the pathogen, in the fungal compared to the control treatment (wounding, no fungus) and the reference (healthy sample collected at the time of wounding and inoculation), at two different host locations, and at different sampling times (3 and 43 days).
The results of this work suggested that different metabolic processes occur in bark and sapwood after wounding and fungal inoculation, compared to healthy samples collected before treatment: in bark, greater peaks were elicited in the aromatic region, whereas in sapwood lower concentrations of all metabolites were observed in inoculated samples, compared to healthy samples. Multivariate statistical analysis carried out with principal component analysis showed highly significant effects of reference, location, and time, and significant effects of clone and fungus. Differences between clones were apparent in sapwood but not in bark, and were due to peaks in the aliphatic region. Over time, in bark there was a decrease in carbohydrate peaks, followed by an increase in aliphatic and aromatic peaks. Sapwood, by contrast, showed a decrease in all peaks, followed by an increase in carbohydrate and aliphatic peaks. Changes in carbohydrate levels were observed within the lesion compared to more distal location in both bark and sapwood.
(2) Expression profiles of a range of defence-related genes, including phenylalanine ammonia lyase (PAL), cinnamoyl-CoA reductase (CCR1), hydroxycinnamoyl CoA shikimate / quinate hydroxycinnamoyltransferase (HCT1) and cinnamyl alcohol dehydrogenase (CAD) of the phenylpropanoid pathway, peroxidase (PaPX3), and a class IV chitinase (PaCHI4), with a-tubulin used as endogenous control, were investigated in bark and sapwood of Picea sitchensis clones showing similar susceptibility to Heterobasidion annosum s.s. 3 days after wounding and artificial inoculation with the pathogen. Constitutive levels of expression were compared with levels induced after treatment at two locations (at the site of inoculation and distally, 1 cm away from the inoculation site). In addition, lignin and soluble and cell wall bound phenolic compounds were extracted from the distal zone only, after inoculation.
Compared to constitutive levels, metabolic processes differed between bark and sapwood: after treatment, in bark all genes were up-regulated, whereas in sapwood all genes except for PaPX3 and PaCHI4 were downregulated. In bark all genes investigated, except for CAD, were expressed at greater levels at the site of inoculation than in the distal zone, whereas in sapwood PAL, CCR1, HCT1 and CAD were present at lower levels around the site of inoculation than in the distal zone. Furthermore, compared to wounding only, inoculation with H. annosum triggered significantly different CAD, PaPX3, and PaCHI4 levels in bark but not in sapwood, indicating differences in the defence response between these two tissues. Different quantities of lignin and soluble and cell wall bound phenolic compounds were found in bark and sapwood. In bark, wounding and inoculation led to different concentrations of cell wall bound phenolic compounds (unknown2, unknown3, coniferin, astringin, taxifolin, piceid, and isorhapontin) compared to healthy control samples, whereas in sapwood, concentrations did not differ following treatment.
These results indicated that bark of Sitka spruce has a stronger and earlier response to wounding and pathogen inoculation than sapwood.
Potential impact
These results are of high significance in the forest industry throughout the northern hemisphere. The work has demonstrated the different techniques can be utilised to detect levels of susceptibility / resistance to Heterobasidion annosum. Compared with long-term susceptibility trials, these techniques provide rapid methods for susceptibility testing in newly available spruce clones so that plants showing greater resistance to the pathogen can be used on forest sites conducive to disease development. Further testing is required to fully corroborate the methods developed in this work against those used in younger plants in the earlier work (Bodles et al., 2006, 2007; Woodward et al., 2007). It is also important to note that, following confirmatory testing, the results obtained in this work will be equally applicable to other important species of conifer in the Pinaceae. Securing forest productivity in this manner has extensive socio-economic impacts in terms of retaining rewarding employment in the rural sector, improving yields from European forests and increasing quality timber output, thereby improving competitiveness of European forestry industries in the world markets.
Contacts:
Dr Giuliana Deflorio and Dr Steve Woodward
Institute of Biological and Environmental Sciences
University of Aberdeen
Scotland, United Kingdom
Professor Marcel Jaspars
Department of Chemistry
University of Aberdeen
Scotland, United Kingdom
Dr Carl Gunnar Fossdal
Norwegian Institute of Forest and Landscape
Ås, Norway
email: s.woodward@abdn.ac.uk
Tel.: +44-122-4272669
References:
- Bodles, W. J. A., Fossdal, C. G. and Woodward, S. (2006). Multiplex real-time PCR detection of pathogen colonisation in the bark and wood of Picea sitchensis clones with different levels of resistance to Heterobasidion annosum. Tree Physiology 26: 775-782.
- Bodles, W. J. A., Beckett, E. and Woodward, S. (2007) Responses of Sitka spruce from different origins to inoculation with Heterobasidion annosum: Fungal growth, lesion development resin duct development and lignosuberised boundary zone formation. Forest Pathology 37:174-186.
- Woodward, S., Bianchi, S., Bodles, W. J. A., Beckett, E. and Michelozzi, M. (2007). Physical and chemical responses of Sitka spruce (Picea sitchensis) clones to colonisation by Heterobasidion annosum as potential markers for relative host susceptibility. Tree Physiology 27:1701-1710.