MONITORING OF COLD STRESS IN TREES BY ACOUSTIC EMISSION TECHNIQUE, THANKS TO WAVEFORM FEATURE ANALYSIS – ACOUFREEZE

Xylem embolism is a limiting factor for plant survival and distribution. Two major factors can induce embolism in the xylem of plants: drought and freeze stress.
Several methods are available to measure embolism, but these methods are destructive and do not allow tracking embolism in natura.
The acoustic method allows monitoring during freeze-thaw cycles on conifers. The main objective of this project was to test acoustic method on Angiosperms (10 species) by comparing the frost temperature effect.
For this, the minimal temperature of the frost cycle correlated positively with induced PLC, whereby species with wider conduits(hydraulic diameter) showed higher freeze-thaw-induced PLC. Ultrasonic activity started with the onset of freezing and increased with decreasing subzero temperatures, whereas no UE were recorded during thawing.
The temperature at which 50% of UE were reached varied across species and was in relationship with the 50% of PLC. These findings indicate that temperatures during freezing are of relevance for bubble formation and air seeding.

Our study shows that a new non-invasive methods, based on the detection and analysis of ultrasonic signals transmitted by tree after freeze/thaw event can follow the vulnerability of xylem vessels and living cells of different species
Our study shows also species-specific cavitation thresholds are reached during freezing due to the temperature-dependent decrease of ice water potential, while bubble expansion and its resulting embolism occur during thawing. Ultrasonic emission analysis enabled new insights into the complex process of xylem freezing and might be used to monitor ice propagation in a whole plant in natura in relation with stress intensities and physiology

We report recent advances on how UE detection could provide insights into:
(i) the ice nucleation and its propagation (Charrier et al., 2015, Charra-Vaskou et al., in press),
(ii) the ice distribution within xylem (Charrier et al., 2014a),
(iii) the loss of hydraulic conductivity after a freeze-thaw cycle (Charrier et al., 2014b),
(iv) the damages generated on living cells (Kasuga et al., 2015 [8]).

Results indicate that cavitation events are generated at the ice front leading to UAE. Species-specific cavitation thresholds are reached during freezing due to the temperature-dependent decrease of ice water potential, while bubble expansion and its resulting PLC occur during thawing. This very low water potential at the water-ice interface is crucial because it can cause both an irreversible plasmolysis of the living cells in wood and bark (water flow) and embolism of xylem vessels. Contrarily to drought-induced embolism, UAE analysis during freeze-thaw cycles allow to distinguish between cavitation and embolism stages, according to the freezing and thawing processes, respectively. Ultrasonic emission analysis enabled new insights into the complex process of xylem freezing and might be used to monitor ice propagation in a whole plant in natura in relation with stress intensities and physiology.

The consortium of this project allowed obtaining a great academic production (10 peer-reviewed publications) and quality (IFMean = 5.895) in just 3 ½ years. These very good results are related to the complementarity of the two teams had worked together during two previous Huber Curien projects (Project Amadeus 2004-2005 No. 11/2004. Freeze-thaw induced embolism in conifers 2009- Amadeus Project 2010, No. 19440YE: Resistance to water and freezing stress: intraspecific variation in conifers and angiosperms) and with the industrial partner, world leader in acoustic techniques used in this project. Note here that the exchange of our postdocs (Dr Katline Charra-Vaskou: PhD from the University of Innsbruck, recruited in Post-Doc on this project in Clermont-Ferrand and Dr Guillaume Charrier, PhD of the University Blaise Pascal in Clermont-Ferrand and recruited in Post-Doc at the University of Innsbruck) has been a major point of the success of this project.

Climate change will affect the sustainability of forest ecosystems, which will result in strong ecological and economical repercussions. Mean temperature is expected to increase during the next century, which will lead to a reduction in frequency but not in intensity of frost events. Under these conditions, frost damages are very likely to increase, because paradoxically, the ability to withstand a deep frost event acquired gradually falls under the influence of lower temperatures. A major challenge for research is to provide relevant and operational criteria in order to identify genotypes more resistant to climatic hazards.

Winter freezing tolerance is one of the key factors limiting survival and distribution of plants in many ecosystems. Freezing survival of plants involves tolerance of living tissues and of the nonliving water transport system (xylem). For living cells, damage and subsequent death may occur when intracellular water freezes or when cells dehydrate due to extracellular freezing of the sap. Plant xylem can be affected by freeze-thaw induced embolism as embolised conduits do not contribute to water transport. Before onset of the vegetation period, water transport has to be restored in all species by the development of new functional conduits and/or the refilling through active mechanisms, whereby living cells (cambium or vessel-associated cells) play a key role. Frost stress monitoring can be achieved with the electrolyte leakage test (LT50) for cells lyses and with quantification of the percent loss in hydraulic conductivity (PLC) for winter embolism. These two methods are rather laborious and time consuming. Recently, the Austrian Partner developed a new method for conifers to quantify drought induced PLC by analysis of waveform features of Acoustic Emission (AE) energy. We see a high potential in the AE method for the analysis of frost damage both, in laboratory resistance tests and in field measurements on intact plants.

The proposed project will focus on the analysis of frost resistance and monitoring of frost stress in a selected set of economically important European tree species. The study will be based on the AE technique with real time detection and wave form analysis of AE events, whereby wave form features should allow discriminating damage events in living cells and non-living tissues and the pinpointing of periods of severe frost stress online. A portable prototype for AE monitoring developed by the industrial partner will be used for monitoring of frost stress in natura. Based on this new methodical possibilities, (i) resistance to frost damage of selected species and a characterisation of AE signals related to the species’ anatomy, (ii) visualisation of ice formation and water flow in freezing stems and (iii) frost damage in living cells and the xylem of trees growing at the alpine timberline as well as resistance characteristics of alpine tree species will be analysed. The promising combination of experimental and field studies is based on the cooperation of three partners complementing one another: Partner 1 (INRA, France) has long term experience in analysis of plant frost resistance, partner 2 (University Innsbruck, Austria) is a specialist for winter stress in alpine trees and ultrasonic measurements on trees and the industrial partner 3 (Mistras Group SA, France) will be responsible for technical developments.

The new methodical approach and acquired data set on frost effects will help to improve future forest monitoring and management strategies and to optimise selection of forest trees and cultivars with high frost tolerance. It will enable new insights into the mechanisms of frost damage and resistance.