D2.1 – Common protocols and reference standards for selected traits and species

Project no. 284181

TREES4FUTURE - Designing Trees for the Future

Start date of project: 1 November 2011 Duration of project: 4 years Call: FP7-INFRASTRUCTURES-2011-1 Theme: INFRA-2011.1.1.13 [Research infrastructures for forestry research] Funding Scheme: Combination of CP & CSA

D2.1 – Common protocols and reference standards for selected traits and species

Description: Common protocols and reference standards for selected traits and species: Technical cards are drawn up holding common protocols and reference standards at a species or at a trait level, as appropriate, and will be made publicly available to the scientific community.

Due date of deliverable: M42 Actual submission date: M42 Organisation name of lead contractor for this deliverable: vTI (now THÜNEN) WP2 Leader: Bart de Cuyper (VLAGEW) Contributor(s): INRA (J-C Bastien, L Paques, A Dowkiw, A Ducousso, F Santi), ALTERRA (SMG de Vries, J Buiteveld), CIDTG (M Touza Vazquez), CRA (F Ducci, MC Monteverdi, R Proietti), FCBA (L Harvengt, D Micheau), IBL (J Kowalczyk, M Rzonca), ICAS (EN Chesnoiu), INIA (R Alia, E Notivol), Metla (now LUKE) (E. Beuker), VLAGEW (B de Cuyper, M Steenackers), vTI (now THÜNEN) (M Liesebach, V Schneck)

Dissemination level PU Public (must be available on the website) X PP Restricted to other programme participants (including the Commission Services) RE Restricted to a group specified by the consortium (including the Commission Services) CO Confidential, only for members of the consortium (including the Commission Services)

1 D2.1 – Common protocols and reference standards for selected traits and species

Table of contents

1. Summary 4 2. Context 5 3. Species oriented common protocols 6 3.1 Broadleaves 6 3.11 Beech specific protocols 6 3.111 Flushing 6 3.12 Oaks specific protocols 12 3.121 Flushing 12 3.13 Ash specific protocols 16 3.131 Flushing 16 3.14 Poplars specific protocols 19 3.141 Phenology 19 3.142 Melampsora laric-populina 23 3.143 Marsonia brunea 27 3.144 Xanthomonas 28 3.15 Wild cherry specific protocols 30 3.151 Phenology 30 3.152 Wood color 36 3.16 Broadleaves non-species specific protocols 39 3.161 Straightness 39 3.162 Forking 41 3.163 Branch habit 42 3.2 Conifers 45 3.21 Norway spruce protocols 45 3.211 Flushing 45 3.22 Douglas-fir specific protocols 47 3.221 Phenology 47 3.222 Frost hardiness 51 3.223 Needle cast 52 3.23 Scots pine specific protocols 54 3.231 Lophodermium needle cast 54 3.232 Pine twisting rust 56 3.24 Mediterranean pines 58 3.241 Drought 58 3.242 Flowering 58 3.243 Polycyclim 59 3.244 Bark thickness 60 3.25 Larch specific protocols 62 3.251 Phenology 62 3.252 Canker 64 3.253 “Drought” cracks 66 3.26 Conifers non-species specific protocols 68 3.261 Stem straightness 68 3.262 Basal sweep 71

2 D2.1 – Common protocols and reference standards for selected traits and species

3.263 Forking 72 3.264 Branching habit 73 4. Traits oriented common protocols 77 4.1 Wood quality 77 4.11 General overview if available techniques and equipment 78 4.12 Acoustic tools 80 4.13 Near Infrared Spectroscopy (NIR) 81 4.14 Innovation and future trends 82 4.2 Drought resistance 87 4.21 Methodology and Evaluation System 88 4.22 Preliminary results 88 4.23 Conclusions 93 5. Reference genotypes 100

3 D2.1 – Common protocols and reference standards for selected traits and species

1. Summary

Systematic provenance and progeny trials have been established for more than 100 years. Many measurements and assessments were carried out within these trials. Standards were defined for the traditional measurements like height and diameter (dbh). Numerous scoring schemes were developed for other traits. This makes it difficult or not feasible to compare results between institutions, but also between test series and single trials. Within the FP7 project TREES4FUTURE, this topic is addressed in “Work package 2 – Creating common standards and protocols”. The deliverable of “Task 2.1 – Development of common protocols and selection of reference genotypes” presents species specific, and non-species specific common protocols for five broadleaves (species or species groups), and five conifers (species and species groups), respectively. The status and development of the assessment of wood characteristics and drought are given. Furthermore, the deliverable presents a concept and a selection of reference genotypes.

Key words: assessment, phenology, stem form, disease, wood quality, drought, reference genotype

4 D2.1 – Common protocols and reference standards for selected traits and species

2. Context

Many traits are of interest to forest tree breeders to distinguish genetic units, and to describe genetic variation and the evaluation of genotypes along their breeding programs. A lot of scoring systems have been developed by all tree breeders. This makes results between institutions but also between test series and single trials difficult or unfeasible to compare. Common protocols were used for a limited number of trial series. However, this is mostly infrequent. Criteria for common assessments methods are: acceptable, simple, easy to handle (fast), low-cost, defined for a specific part in the life cycle. The starting point for Task 2.1 was an inventory of existing national assessment schemes. The inventory could be assembled from the results of the FP6 project TREEBREEDEX (PÂQUES 2009, DUCCI et al. 2012). The inventory was essential because the tree species beech and oaks were not included in TREEBREEDEX. Further protocols drawn up from the literature were added. During the workshops a selection was taken and common protocols identified. The task involves a large number of participants, each with their own species of interest, leading to a high number of species x traits combinations. Moreover, common protocols are usually not transferable from one species to another. Not all species x traits combinations can be tackled within the time span of the project, so priorities will have to be set regarding the species as well as regarding the traits to be assessed. A selection was made of six target species (beech, ash, wild cherry, Norway spruce, Douglas fir, Scots pine) and four species groups (oaks, poplars, Mediterranean pines, larches) of high interest from a European point of view (Chapter 3). The number of common protocols varied from one each of the beech and oak species to seven for the species group poplars. Furthermore common protocols were identified for broadleaves and conifers. For each species/species group a core group was created and a responsible core group leader determined. Specific input was provided at the trait level for the assessment of wood characteristics and drought based on the results of questionnaires (Chapter 4). Furthermore, the concept of reference genotypes and a selection of extreme reference genotypes with low genotype-environmental-interaction are presented (Chapter 5).

References

PÂQUES L (comp.), 2009: European survey on assessment methods of morphological traits. Treebreedex Doc. 916: 53 pp.

DUCCI F, DE CUYPER B, PAQUES L, PROIETTI R, WOLF H (comp.), 2012: Reference protocols for assessment of trait and reference genotypes to be used as standards in international research projects. CRA SEL, Arezzo, Italy: 82 pp.

5 D2.1 – Common protocols and reference standards for selected traits and species

3. Species oriented common protocols

3.1 Broadleaves

3.11 Beech specific protocols

Core group: Mirko Liesebach (vTI now THÜNEN), Sven M.G. de Vries (ALTERRA ), Marek Rzonca (IBL), Ecaterina Nicoleta Chesnoiu (ICAS), Ricardo Alia (INIA), Alexis Ducousso (INRA)

3.111 Flushing (bud break)

Definition A specific trait of beech (Fagus sylvatica L.) is flushing (bud break), which is under strong genetic control. Results from provenance trials show large differences in leaves’ flushing time in spring. Generally, provenances from the eastern and south-eastern part of the distribution area, as well as provenances from high elevations, require a smaller heat sum for flushing and thus flush earlier. Provenances from the western part of the distribution and from low elevations where late frosts occur require a higher heat sum and flush late. Depending on the temperature development during spring, the time differences between the earliest and the latest individuals to flush in a set of provenances in a trial can be 4 to 6 weeks. Between the different years and over a number of sites, certain stability was found in the ranking of flushing time in the provenances analysed. The phenology of flushing is important for damages by late frost. In the context of climate change that becomes more and more important. Damages by late frost can result in yield losses and poor stem forms. Therefore, flushing is a trait which has an indirect economic impact, apart from its adaptive value.

Description of the assessment in general Flushing will be recorded for each tree separately. Observations on beech show that there is no rule whether the terminal bud or other buds starts flushing. Experience shows that in younger plants a stage is reached when approximately 3 buds show the specific characteristics. Flushing can easily be recorded at younger plants up to a height of 2 m (average height of a trial series) For older and large plants it is almost impossible or at least not easy to record single buds, i.e., when the average height of a trial site is above 2 m. In this case a stage is reached when 50 % of the crown shows the characteristics of a specific stage. This is according to the BBCH recommendations.

6 D2.1 – Common protocols and reference standards for selected traits and species

Depending on the scientific question and the financial resources, records will be taken (1) once, all trees at a time (each stage) or (2) periodically (2.1) each stage, all trees at each assessment, (2.2) one specific stage, only trees recently flushed at each assessment. When the assessment should only be carried out once, the timing is important. The good variation between the genetic units is around a mean value of the scoring schema for a site. The assessment has to be carried out uninterrupted and should not last more than one day. A replication should be assessed without a break. The assessment of flushing should first be done at the beginning of the second vegetation period in the field. For older plants, the assessment is only to get an impression whether a genetic unit is flushing early or late.

Scoring schema a) For trees up to a mean height of 2 m of a trial series a 5-step-scale is recommended: Scale values Text 1 dormant winter bud 2 buds expanding 3 bud-burst (first green is visible) 4 leaves are flushing 5 leaves are fully expanded

Stage 1

7 D2.1 – Common protocols and reference standards for selected traits and species

Stage 2

Stage 3

Stage 4

8 D2.1 – Common protocols and reference standards for selected traits and species

Stage 5

Photos: ALTERRA (left), M. LIESEBACH (right) b) For trees above a mean height of 2 m, a 3-step-scale is recommended: Scale values Text 1 (1) dormant winter bud (late flushing) 2 (3) bud-burst (first green is visible) (intermediate) 3 (5) leaves are fully expanded (early flushing) (numbers in brackets correspond to the scale mentioned under 3.1)

1 2 3

Photos: ALTERRA

Pros and cons of the described protocols a) Pros There is good experience in using the protocols. Genetic units can be distinguished nicely coming at the right time for the assessment.

9 D2.1 – Common protocols and reference standards for selected traits and species

Several assessment schemas were used in the past. The results of existing accessments can only be converted backwards with losses in a few steps (see Table 3.11.1) not vice versa. The data can be analysed as a quantitative trait by adjusting a Weibull function and can be easily compared among sites and recording dates.

Table 3.11.1: Assessment schemas used for scoring flushing at beech trials 3-step 5-step 6-step 7 step 1= late flushing 1= dormant winter bud 1= dormant winter bud 1= dormant winter bud 2= buds expanding 2= buds swollen 2= buds swollen 3= bud-burst (first green is 3= first green is visible 3= first green is visible 2= intermediate visible) 4= leaves begin to 4= leaves begin to appear 4= leaves are flushing appear, individually 5= individually visible visible folded folded 3= early flushing 5= leaves are fully expanded 5= leaves unfolded, fan- 6= leaves unfolded, shaped fanshaped 6= leaves unfolded, 7= leaves unfolded, bright bright b) Cons It is not always easy to catch the right time to have a good differentiation between the genetic units in flushing.  Difficulties in choosing proper term of assessment,  possible subjective influence,  requires trained staff.

References Alterra, 2005 (in Dutch): INS-CGN-FG-076 Uitloopstadia Fagus sylvatica.

LIESEBACH M, 2012 (in German): Phenotypical traits and variability of six European beech (Fagus sylvatica L.) provenances on a test site in Schleswig-Holstein. Landbauforschung - Journal of Applied Research in Agriculture and Forestry 62 (4): 179-192.

MADSEN SF, 1995: International beech provenance experiment 1983-1985. Analysis of the Danish th member of the 1983 Series. Genetics and Silviculture of Beech, Proceedings from the 5 Beech Symposium of the IUFRO Project Group P1. 10-100, 19-24 September, 1994, Mogenstrup, Denmark, 35-44 + 288

MALAISSE F, 1964: Contribution à l’étude des hêtraies d’Europe occidendentals. Note 4: Quelques observations phénologiques de h1etraies en 1963. Bull. Soc. r. Bot. Belg. 97: 85-97.

MATRAS J, et al., 2005 (in Polish): Growth at the young age and genetic variability of European beech (Fagus sylvatica L.) populations in Poland. Scientific report for years 2001-2005 with recommendations for practise, Warsaw, 105 p.

ROBSON TM, ALIA R, BOZIC G, CLARK J, FORSTEUTER M, GÖMÖRY D, LIESEBACH M, MERTENS P, RASZTOVITS E, ZITOVÁ M, VON WÜHLISCH G, 2011: The timing of leaf flush in European beech (Fagus sylvatica L.) saplings. Monografias INIA: Serie forestal, vol. 22: 61-80.

10 D2.1 – Common protocols and reference standards for selected traits and species

SCHÜLER S, LIESEBACH M, 2015: Latitudinal population transfer reduces temperature sum requirements for bud burst of European beech. Plant Ecol 216: 111-122.

SCHÜLER S; LIESEBACH M, WÜHLISCH G VON, 2012 (in German): Variation in flushing time of European beech: effects of provenance, year and environment. Landbauforschung - Journal of Applied Research in Agriculture and Forestry 62 (4): 211-219.

11 D2.1 – Common protocols and reference standards for selected traits and species

3.12 Oak (Quercus spp.) specific protocols

Core group: Marek Rzońca (IBL), Sven M.G. de Vries (ALTERRA), Ecaterina Nicoleta Chesnoiu (ICAS), Alexis Ducousso (INRA), Mirko Liesebach (vTI now THÜNEN)

3.121 Flushing (bud break)

Definition Tree phenology changes seasonally due to both climate and endogenous rhythms. A specific trait for oak species is flushing, (bud break). Bud break is the first visible step in the annual cycle of trees growth. In oaks species, early wood, including large vessels and a part of the annual radial stem growth is achieved before leaf expansion in spring. Oaks have two types of flushing, early and late. Long term observations show about 2-3 week differences between these types. In the southern range (northern Italy) flushing starts on about 20 April, in the northern range (southern Scandinavia) on 20 May (for Quercus petraea, Q. robur). Spring flushing dynamics and terms of dormancy are largely genetically determined. The variability of flushing is important for damages by late frost, causes in yield losses and poor stem forms. Consequently, flushing is a specific visible trait, which affects the oaks’ adaptive potential and economic value.

Description of the assessment in general The assessment of oak flushing is similar to beech. Bud break should be assessed separately for each tree in the field. Due to size of observed material (height of tree) flushing can be more easily recorded for younger tree, especially those up to 2 m of height. Taller trees should be assessed with another scale. In this case is not possible to observe single buds. The BBCH recommendation states that when 50 % of the crown achieves the specific characteristics, the stage is reached. The issue of the date of assessment, it should be carried out in several ways:  One time (all trees in one date)  Periodically - In each stage (all trees at severals date and each assessment) - In only one specific stage (only trees recently flushed at each assessment) When the assessment is carried out, the most important factors is to check for a good time for observation. If the assessment starts too early, buds are not variably developed and cannot be observed at all stages of the scale. The same problem appears when assessment starts too late. The good variation among trees is about a mean value of scoring schema for an each field trial.

12 D2.1 – Common protocols and reference standards for selected traits and species

Scoring scheme a) For trees up to a mean height of 2 m, a 5-step-scale is recommended: Scale values Text 1 dormant winter bud 2 buds expanding 3 bud-burst (first green is visible) 4 leaves are flushing 5 leaves are fully expanded

1 2 3 4 5

Photos: MLUV b) For trees above a mean height of 2 m. a 3-steps-scale is recommended: Scale values Text 1 (1) dormant winter bud (late flushing) 2 (3) bud-burst (first green is visible) (intermediate) 3 (5) leaves are fully expanded (early flushing) numbers in brackets correspond to the scale mentioned under a)

1 2 3

13 D2.1 – Common protocols and reference standards for selected traits and species

1 2 3

Photos: E. N. CHESNOIU

Pros and cons of the described protocols a) Pros  There is a lot of good experience in using this protocol, the scale is in practice. The results of existing accessments can only be converted backwards with losses in a few steps (see Table 3.12.1) not vice versa.  it is possible to aggregate some steps for comparing assessments (Table 3.12.1),  this scheme has been discussed among a broad range of foresters (e.g., Treebreedex).

Table 3.12.1: Assessment schemas used for scoring flushing at oak field trials 3-step 5-step 5-step BBCH 6-step 8-step 1= late 1= dormant 1=00= buds are closed 1= dormant 0=dormant winter flushing winter bud and covered in brown winter bud buds scales 2= buds 2=01= the majority of 2= buds 1=buds swollen expanding leaf buds have started swollen to expand 3= bud-burst 3=03= the majority of 3= buds begin 2=buds swollen and (first green is leaf buds have finished to open elongated 2= visible) expanding and are 4= individual 3=buds burst, first intermediate ready to open small leaves green is visible 4= leaves are 4=07= start of budburst are visible 4=buds begin to flushing and leaf growth open, leaves gathered into a bunch 3= early 5= leaves are 5=09= the tips of the 5= leaves are 5=individual small flushing fully expanded first leaves are clearly big, but still leaves are visible visible folded 6=leaves are big, but still folded, main shoot starts to grow 6= leaves 7=leaves unfolded unfolded and and smooth, main smooth shoot is growing

14 D2.1 – Common protocols and reference standards for selected traits and species

b) Cons  It is not always easy to catch the right time to have a good differentiation between the genetic units in flushing.  Difficulties in choosing proper term of assessment,  possible subjective influence,  requires trained staff.

References

BREDA N, GRANIER A, BARATAUD F, MOYNE C, 1995: Soil water dynamics in an oak stand. Part I: soil moisture, water potentials and water uptake by roots. Plant and Soil 172: 17–27.

MARKIEWICZ P et al., 2006 (in Polish): Variability of the growth and quality of pedunculate oak (Quercus robur L.) populations and families in Poland. Scientific report for years 2002-2006 with recommendations for practise, Sekocin Stary, 45 p.

SECONDAT N, 2008: Study of the phenology of the sessile oak (Quercus petraea). Masters project, University of Bordeaux, 13 p. MLUV [Ministerium für Ländlicher Entwicklung, Umwelt und Verbraucherschutz des Landes Brandenburg, Landeskompetenzzentrum Forst Eberswalde] (ed.) 2009 (in German): In the food- steps of climate change: Phenology of forest trees. Leaflet.

15 D2.1 – Common protocols and reference standards for selected traits and species

3.13 Ash specific protocols

Core group: Joukje Buiteveld (ALTERRA), Arnaud Dowkiw (INRA), Ecaterina Nicoleta Chesnoiu (ICAS), Bart de Cuyper (VLAGEW)

3.131 Flushing (bud burst)

Bud burst is the period of the initiation of growth from a bud till elongated shoots with expanded leaves. The trait is under strong genetic control (high heritability) in ash and is influenced by environmental factors and tree health conditions. The most important environmental factors influencing the timing of flushing are spring temperature and winter chilling. Data from provenance trials indicate large differences in flushing time between provenances. In general, northern and western provenances flush later than southern and eastern ones. Also, provenances from high elevations flush later than the ones from low elevations. There is a strong positive correlation between flushing dates between the years. Flushing is an important adaptive trait. Early flushing provenances/trees are prone to damage from late spring frost. This damage may reduce the growth of the tree and affect the tree’s fitness and stem form. There is evidence from more recent studies that flushing is correlated with susceptibility to ash dieback, and that late-flushing trees are more susceptible to infection by fungus.

Description of the assessment in general Evaluation of bud burst dynamics is based on observations over a period of time with a scoring system describing the different stages for the development of the bud and unfolding of the leaves. It will be assessed on single trees. In principle the terminal bud is scored in young trees up to a height of 5m (average height of a trial series). For older trees (height above 5m) it is difficult to score the single terminal buds. Here a stage is reached when 50% of the crown meets the described criteria of a specific developmental stage (conform BBCH recommendations). Depending on the research objectives/available resources different ways of assessment are possible: • Observation only once in spring. The time is chosen so that a maximum variation of the stages occurs in the population of trees to be studied. • (Julian) day when a particular developmental stage is reached. • Observation of every tree at steady intervals (of 3 days or a week) during the whole flushing period (gives better insight in the dynamical aspects of bud burst behaviour during the whole observation period). Each tree is preferably scored on a single day.

16 D2.1 – Common protocols and reference standards for selected traits and species

Scoring scheme For young trees of a trial site up to 5 m, a 5-point scale is recommended: Scale values Text 1 dormant bud 2 swelling of bud, slight greening of bud scales 3 buds begin to burst, first green visible 4 bud burst, petioles of leaves visible, no lengthening of twig 5 bud burst, petioles of leaves visible, twig has started lengthening, leaves are fully expanded

1 2 3 4 5 Photos: INRA

For older trees (above 5 m) a 3-point scale is recommended: Scale values Description 1 (1) dormant bud 2 (3) buds begin to burst, first green visible 3 (5) bud burst, petioles of leaves visible, twig has started lengthening, leaves are fully expanded (numbers in brackets correspond to the 5-step scale for smaller plants)

17 D2.1 – Common protocols and reference standards for selected traits and species

1 2 3 Photos: INRA

Pros and Cons a) Pros The scoring system covers the total range of developmental stages described, including shoot elongation. It is quick and simple. b) Cons Timing of observation is important to record maximum variation in the trial.

References

CUNDALL EP, CAHALAN CM, CONNOLLY T (2003) Early results of ash (Fraxinus excelsior L.) provenance trials at sites in England and Wales. Forestry 76: 385–400.

DOUGLAS GC, PLIURA A, DUFOUR J, MERTENS P, JACQUES D, FERNANDEZ-MANJARES J, BUITEVELD J, PARNUTA G, TUDOROIU M, CURNEL Y, THOMASSET M, JENSEN V, KNUDSEN M, FOFFOVÁ E, CHANDELIER A, STEENACKERS M, 2013: Common Ash (FRAXINUS EXCELSIOR L.) In: L.E. Pâques (ed.), Forest Tree Breeding in Europe: Current State-of- the-Art and Perspectives, Managing Forest Ecosystems 25, DOI 10.1007/978-94- 007-6146-9_9.

VITASSE Y, DELZON S, DUFRÊNE E, PONTAILLER J-Y, LOUVET J-M, KREMER A, MICHALET R, 2009: Leaf phenology sensitivity to temperature in European trees: Do within-species populations exhibit similar responses? Agricultural and forest meteorology 149: 735 – 744.

BAKYS R, VASAITIS R, SKOVSGAARD JP, 2013: Patterns and Severity of Crown Dieback in Young Even-Aged Stands of European Ash (FRAXINUS EXCELSIOR L.) in Relation to Stand Density, Bud Flushing Phenotype and Season. Plant Protection Science Vol. 49 (3): 120-126.

JOUVE L, JACQUES D, DOUGLAS GC, HOFFMANN L, HAUSMAN J-F, 2007: Biochemical characterization of early and late bud flushing in common ash (Fraxinus excelsior L.) Plant Science 172: 962–969.

18 D2.1 – Common protocols and reference standards for selected traits and species

3.14 Poplars specific protocols

Core group: Marijke Steenackers (VLAGEW), Sven MG de Vries (ALTERRA), Catherine Bastien (INRA), Alain Berthelot (FCBA) Lorenzo Vietto, Fulvio Ducci (CRA), Volker Schneck, Mirko Liesebach (vTI now THÜNEN)

3.141 Phenology

3.141.1 Bud burst In springtime, when air temperatures rise to optimal levels, the buds swell and new leaf tissue pushes out of the protective scales. This process is called bud burst or bud break. The objective of the assessment of the earliness of vegetative budbreak is an indirect selection for resistance to spring late frosts.

Description of the assessment Bud break is evaluated on terminal shoot at two or three years in nursery test with a 6 classes score. The most important stage associated to frost damages is stage 3 (see scoring scheme). Good correlation between nursery stage and adult stage in plantation have been observed. Bud break earliness is assessed at two year old trees in nursery test on terminal buds of main stem or codominant shoots (coppice). When done at only one date, the assessment of bud break with this 6 classes score is optimum when about 50 % of trees could be scored 3 or more. Optimal evaluation of bud break earliness is obtained with an average score collected on three successive dates (date 1: 25 % of trees have reached score 3 or more, date 2 : 50 % of trees have reached score 3 or more, date 3 : 75 % of trees have reached score 3 or more.

Scoring scheme Scale values Text 1 Dormant bud completely enveloped by the scales (= perulae) 2 Bud swelling with scales slightly diverging showing a narrow yellow margin; presence of one or more droplets of balsam 3 Bud sprouting, with tips of the small leaves emerging out of the scales 4 Buds completely opened with leaves still clustered together; scales still present 5 Leaves diverging with their blades sill rolled up; scales may be present or absent 6 Leaves completely unfolded (but smaller in size than mature ones); lengthening of the axis of the shoot evident; scales absent

19 D2.1 – Common protocols and reference standards for selected traits and species

1 2 3 4 5 6

Pro and con This protocol is suitable to measure quickly a large number of trees.

Reference clones Budflush phenology Standard clones Early budflush Flevo, I-214, Soligo Medium budflush Alcinde,Catherine aanvullen Late budflush Koster, Dorskamp, Blanc du Poitou, Triplo

References

CASTELLANI E, FRECCERO V, LAPIETRA G, 1967: Proposta di una scala di differenziazione delle gemme fogliari del pioppo utile per gli interventi antiparassitari. Giorn Bot Ital 1967, 101: 355-360.

TUROK J, LEFÈVRE F, CAGELLI L, DE VRIES SMG, (compilers), 1996: Populus nigra Network. Report of the second meeting, 10 – 12 September 1995, Casale Monferrato, Italy. Rome, Italy: International PlantGenetic Resources Institute, 26 pp.

3.141.2 Bud set Definition The objective of the assessment bud set lateness is an indirect selection for resistance to early frosts in autumn. Date of bud set is evaluated on terminal shoot at two years in nursery test with the following 7-classes-score (protocols B or C). The most important stage is stage 1,5; stage 2,5 correspond to cessation of elongation. Length of photoperiod and average temperature are known to influence jointly date of growth cessation and date of bud set.

20 D2.1 – Common protocols and reference standards for selected traits and species

Description of the assessment Date of bud set is evaluated on terminal shoot at two years in nursery test with the 7- classes-score (protocols B or C) given below. The most important stage is stage 1,5; stage 2,5 correspond to cessation of elongation. Length of photoperiod and average temperature are known to influence jointly date of growth cessation and date of bud set. When done at only one date, the assessment of bud set with this 7-classes-score is optimum when about 50 % of trees could be scored 1.5 or less. Optimal evaluation of date of bud set is obtained with an average score collected on three to five successive dates.

Scoring scheme

Scale values Text 1 (0 in figure) Apical bud red brown No more 2 (0,5) F1 fully stretched comparable to older leaves apical but of older leaves; rolled apical bud of fully closed between green and red leaves 3 (1) F1 fully stretched with ‘juvenile aspect’; F2 comparable to older leaves apical bud not fully closed 4 (1,5) F2 fully stretched transition to bud structure Presence 5 (2) F2 fully stretched no bud structure of rolled 6 (2,5) 2 rolled-up young leaves at equal height leaves 7 (3) >2 rolled-up young leaves

21 D2.1 – Common protocols and reference standards for selected traits and species

22 D2.1 – Common protocols and reference standards for selected traits and species

Pro and con These protocols are suitable to measure quickly a large number of trees.

References

ROHDE A, STORME V, JORGE V, GAUDET M, VITACOLONNA N, FABBRINI F, RUTTINK T,ZAINA G, MARRON N, DILLEN S, 2011: Bud set in poplar - genetic dissection ofa complex trait in natural and hybrid populations. New Phytol 189: 106-121.

RHODE A, BASTIEN C, BOERJAN W, SEAN T, 2011: Temperature signals contribute to the timing of photoperiodic growth cessation and bud set in poplar.Tree Physiol 31 (5): 472-482.

3.142 Melampsora laric-populina

Melampsora larici-populina is the most common and dangerous Eurasian rust, and requires and alternate host „Larix“ to complete its life cycle. Uredinia, telia and basidia are formed on poplar, spermogonia and aecia are formed on larch. Uredinia commonly develop on the lower surface of leaves, giving rise to yellow or necrotic spots and flecks on the upper surface. Severely affected trees are defoliated prematurely, reducing growth potential and predisposing them to environmental stresses and invasion by secondary damaging agents. Early leaf fall also increases the risk for winter injury, dieback and infection by secondary pathogens. The disease is generally controlled by planting tolerant cultivars or clones to the local populations of rust. The potential evolution of new rust pathotypes underscores the critical need of monitoring poplar rust populations.

Description of the assessment in general Different evaluation tests are used to evaluate susceptibility to Melampsora larici-populina: (1) qualitative resistance tests under artificial inoculation with known reference strains; (2) quantitative resistance tests on leaf disks inoculated with known reference strains and (3) susceptibility tests in field experiments under natural inoculation associated to characterization of the pathogen population.

(1) Protocol for qualitative resistance tests under artificial inoculation The objective of qualitative resistance tests is to identify qualitative resistances inherited from P. deltoides and overcome by at least one of the eight known M. larici-populina virulences. Leaf disks collected on disease-free plants are inoculated artificially at high density (40000 spores/ml) with a solution of mono-uredinial strains of known combination of virulences (or pathotype). A minimum of 6 leaf disks sampled from the 5th to the 8th unrolled leaf below the apex are inoculated per tested genotype. After inoculation, leaf disks are maintained in climatic chamber at a temperature between 17°C and 20°C with a 16- hours photoperiod. The response observed 14 days after inoculation is presence/absence of 23 D2.1 – Common protocols and reference standards for selected traits and species

uredinia or any leaf decoloration or any leaf necrosis. A genotype is considered as susceptible to the strain studied when a minimum of one leaf disk presents sporulated uredinia. A genotype is considered as resistant to the strain studied when all the inoculated leaf disks are free of any symptoms. The M. larici-populina strains are chosen in order to represent the eight known virulences. For each inoculation test and each strain, the following differential genotypes are used to confirm the pathotype of the strain used:

List of Mlp virulences for which a Type botanique Differential Clones compatible response oft he differential clone is observed P. × canadensis Robusta 1,2,3,4,5,6,7,8 P. × canadensis Ogy 1 P. candicans Aurora, Grimminge 2 P. × canadensis Brabantica 3 P. x generosa Unal 4 P. × generosa Rap 5 P. deltoides, P. × generosa 87B12, 84B09 6 P. × generosa Beaupré 7 P. ×x generosa Hoogvorst,hazendans 8

(2) Protocol for qualitative resistance tests under artificial inoculation The objective of quantitative resistance tests is to rank poplar genotypes for their ability to limit infection by M. larici-populina in absence of qualitative resistance. The three components of quantitative response associated to pathogen fitness are measured on 3 cm diameter excised leaf disks inoculated with a reference mono-uredinial strain. The leaf disks are floated upside down on distilled water in polycarbonate 6-well cell culture plates. The inoculum suspension concentration was 30 mg/liter : 3 mg urediniospores in 100 ml distilled water added with 0.5 mg agar. Sprayed suspension volume per leaf surface ratio was 20 ml/m2. The disks were kept for 14 days under growth chamber controlled conditions (17 °C, 16 h photoperiod, 30 E.m-2.s-1). The following three components of quantitative resistance are observed on each leaf disk :  the latent period, measured on a half a day basis, which corresponds to the date when the first sporulating uredinia can be observed.  the number of sporulating uredinia 13 days after inoculation

 The average uredinia size score (USscale) 14 days after inoculation. This score uses a 1 to 5 scale defined according to the size variability observed in the experiment. To obtain an objective assessment of uredinia size, 25 leaf disks per score (75 for the whole tests) are sampled for image analysis. Average uredinia surface in mm2 is measured and calculated on all sporulating uredinia of the inoculated leaf disk.

24 D2.1 – Common protocols and reference standards for selected traits and species

(3) Protocol to evaluate susceptibility to Melampsora larici-populina leaf rust under natural infection in field tests Susceptibility to Melampsora larici-populina leaf rust under natural infection can be evaluated by two protocols, dependent on the time of the year: protocol A) the maximum infected leaf score and protocol B) the global sanitary tree score. Protocol A: The maximum infected leaf score quantifies the infection level of the most infected leaf on the terminal shoot. This observation is done quite early, between end of June and mid- July when the infection could be considered as homogeneous in the field experiment. At this time of infection, the most infected leaves are still on the trees. Protocol B: The global sanitary tree score quantifies the damages (infection, necrosis, leaf fall and even plant death) at the plant level. This observation is done late in season, between mid- August and mid-September just before growth cessation.

Pros and cons The protocols are easy to carry out on a large number of trees per time unit (several hundreds per day). The global sanitary tree score could take into account other damages than those directly linked to Melampsora larici-populina infection.

Reference genotypes The following standard clones with known levels of susceptibility to Melampsora larici- populina to populations including the eight known virulences need to be included in field experiments:

Susceptibility level to Mlp in field experiements in Standard clones presence of all virulences Very low susceptible Alcinde, Lena, Bakan, Skado, Soligo, Triplo, Villafranca 87B12, Dvina, I-214, Flevo, Dorskamp, Koster, Fritzi Pauley, Low susceptible Trichobel, Grimminge, Vesten Robusta, Ogy, Brabantica, Unal, 84B09, Blanc du Poitou, I-45/51, Susceptible Raspalje, Vereecken Very susceptible Aurora, Rap, Beaupré, Hoogvorst

It is important to characterize the pathogen population responsible of the natural infection observed in field tests. This characterization is obtained by molecular analysis to confirm the pathogen species and by isolation and pathotype determination of about 100 distinct uredinia sampled on different leaves of the ‘Robusta’ standard clone.

25 D2.1 – Common protocols and reference standards for selected traits and species

Scoring schemes Protocol A: Maximum infected leaf score (1-8); end of July (mid-infection) Source: INRA-FCBA

Scale values Text 8 No uredinia 7 Few difficult to detect 6 Uredinia easy to detect but not joined 5 Joined uredinia covering less than 10 % of the leaf area 4 Joined uredinia covering between 10 % and 25 % of the leaf area 3 Joined uredinia covering between 25 % and 50 % of the leaf area 2 Joined uredinia covering between 50 % and 75 % of the leaf area 1 Joined uredinia covering more than 75 % of the leaf area

6 5 4 3 2 1

Protocol B: global sanitary tree score (1-7); mid-August till mid-September Source: INRA-FCBA, INBO

Scale value Description 7 0 uredinia on the plant (qualitative resistance) 6 Few uredinia hard to detect 5 Many uredinia per leaf but no decoloration neither necrotic zones 4 Many leaves infected but still green. Limited decolorated or necrotic zones, no defoliation 3 Numerous leaves highly infected with decoloration and necrotic areas, significant defoliation but between 1/2 and 1/3 of green leaves on the top of the plant. 2 Most of leaves have decolorations and necrotic areas, important defoliation (30%- 50%), less than ¼ of remaining leaves are still green. 1 Important defoliation (>50 %), no more significant elongation, only few remaining leaves on the top

26 D2.1 – Common protocols and reference standards for selected traits and species

References

DOWKIW A, HUSSON C, FREY P, PINON J, BASTIEN C, 2003: Partial resistance to Melampsora larici-populina leaf rust in hybrid poplars: genetic variability in inoculated excised leaf-disk bioassay and relationship with complete resistance. Phytopathology 93, 421-427.

PINON J, BERTHELOT A, FABRE B, 2011. Comportement des cultivars de peuplier envers la rouille. Revue Forestière française, LXIII, 333-346.

OSTRY M, RAMSTEDT M, NEWCOMBE G, STEENACKERS M, 2014: Diseases of poplars and willows. In: ISEBRANDS JG, RICHARDSON J, (eds.). FAO-book Poplars and Willows: Trees for Society and the Environment, Chapter 8 (pp.443-458)

3.143 Marsonia brunea

Marssonina brunnea is a damaging foliar pathogen of poplar. Severe attacks on susceptible clones can cause premature defoliation, resulting in significant growth reduction, dieback, and predisposition to secondary organisms and abiotic stress. Marssonina brunnea causes dark brown, circular to angular leaf spots that usually measure about 1 mm in diameter. On highly susceptible clones, lens-shaped lesions develop on petioles and current-year shoots. Damage can be especially severe where poplar species and clones are selected without regard to their susceptibility to the disease.

Description of the assessment in general This protocol has been adapted from the international protocol defined in 1975 by the International Poplar Commission and optimized by INRA-FCBA. A special field experiment is established in nursery for a minimum of three years of observation at very high plantation density (0.4 to 0.6 m in the line, 1.5 to 2m between lines) to favor contamination. It is also recommanded to plant at regular spacing a very susceptible reference clone such as Magister Géant or I-214 or Tardif de Champagne. To favor contamination, infected leaves can be attached on the very susceptible reference clone. Between three and five reference clones from the following list are included in the test:

Very susceptible Susceptible reference Resistant/Tolerant Botanic type reference clones clones reference clones P. deltoides Lena Lux Dvina Magister géant, I-214, Robusta, Brenta, Dorskamp, Polargo, P. × canadensis I-45/51, Tardif de Mella, Triplo, Lambro, Lux Champagne Soligo, Blanc du Poitou P. trichocarpa Fritzi Pauley Hazendans, P. × generosa Grimminge, Raspalje Other interspecific hybrids Taro Source: INRA-FCBA

27 D2.1 – Common protocols and reference standards for selected traits and species

Response to contamination is scored twice during the growing season: once early in June and once mid August just before significant leaf fall.

Scoring scheme On the main stem of each plant, all leaves are scored with the following scoring system: Score Description 4 Leaf free of disease 3 Between 1 and 11 acervulae on the leaf 2 Between 11 and 100 acervulae on the leaf 1 More than 100 acervulae on the leaf

Relative frequencies of the 4 leaf scores at an individual tree level are then calculated to estimate genotype susceptibility to Marssonina brunnea.

Pros and cons of the described protocol This protocol is very detailed but not suitable to measure quickly a large number of trees.

3.144 Xanthomonas populi

The bacterium Xanthomonas populi is one of the main poplar pathogens throughout much of Europe. Susceptibility of clones is tested by artificial inoculation on two years old plants in the field. Xanthomonas populi isolates used for inoculation can be obtained from the collection CFBP at INRA-Angers (FR) or from INBO collection, Geraardsbergen (BE). Isolates are reactivated and multiplicated at 20°C in Petri dishes filled with a LPGA medium. The inoculation suspension is adjusted to 108-109 cells per ml just before inoculation. Two or three transversal incisions are made on tree main stem between 0,80 and 1,20m (about 10mm large and 2mm long). 25 µl of the inoculation suspension is deposited on the fresh wound. The inoculation is done in early September, in the period just before closing of the buds. Response to inoculation is measured one and two years after inoculation at each inoculation point thanks to two different traits:  Longitudinal canker extension (mm)  Girdling Index score (0-5):

28 D2.1 – Common protocols and reference standards for selected traits and species

Score Description 6 No symptom 5 canker is developed along less than 20 % of stem circumference 4 canker is developed along between 20 % and 40 % of stem circumference 3 canker is developed along between 40 % and 60 % of stem circumference 2 canker is developed along more than 60 % of stem circumference 1 total girdling of tree stem with associated mortality of the apex

The following reference clones are used for comparison: Very susceptible Botanic type Susceptible to race1 Resistant to race1 to race1 P. x generosa S.6-2 Boelare, Raspalje, Beaupré

P. trichocarpa S.3-31 Fritzi Pauley Blanc du Poitou, P. x canadensis Donk, I-45/51 I-214, Koster Dorskamp

Pros and cons of the described protocol Selection for susceptibility to Xanthomonas populi is a long-term test, as it is done at 1 and 2 years after artificial inoculation on 2-year-old trees.The protocol itself is easy to perform.

References

NÈSME X, STEENACKERS M, STEENACKERS V, PICARD CH, MÉNARD M, RIDÉ S, RIDÉ M, 1993: Differential host- pathogen interactions among clones of poplar and strains of Xanthomonas populi pv.populi. The American Phytopathological Society 84 (1), 101-107.

29 D2.1 – Common protocols and reference standards for selected traits and species

3.15 Wild Cherry specific protocols

Core group: Fulvio Ducci, Roberta Proietti (CRA), Fréderique Santi (INRA), Joukje Buiteveld (ALTERRA), Bart de Cuyper, Marijke Steenackers (VLAGEW)

3.151 Phenology

Definition Phenology is an important aspect of adaption. It is the study of the timing of periodic phenomena such as bud break, flowering, growth initiation, growth cessation, water flows, cambial and physiological activity, especially as related to seasonal changes in temperature, photoperiod, etc. (Nanson 2004). Phenology traits are conditioned by biological and environmental factors (chilling requirements, forcing temperatures, seasonal changes, etc.) necessary for launching the processes, but they are also under strong genetic control. They are important selection criteria as they might condition stem form in the case of stress, frost damage (mostly late frost), but they also affect growth potential in relation to the growth season length. Then they are directly related to the growth and to the tree architecture. Experimental data from already performed surveys show the adaptive meaning of this group of specific traits. Indeed, clones and provenances are strongly influenced in phenology by their origin. Phenology traits are therefore practically relevant indicators of the adaptability and adaptedness of forest trees and are useful to study the effect to global change. A series of assessments on the same individuals over more years allows conclusions about the climate development. It might be useful to identify extreme standard materials which could be used as references in trials to alert for phenology observations. Preferably it should be material shared by as many test sites as possible.

3.151.1 Flushing (bud break) Definition Bud break is the period spanning from dormant bud up to shoot elongation. It is conditioned by internal (genetic) and environmental factors (chilling requirements and forcing temperatures).

Description of the assessment in general A subjective scoring system is commonly used. But in any case, due to the size of trees, different methods need to be adopted depending on whether young or adult trees have to be monitored. In young trees (up to 2.5 m), observation usually concerns the terminal buds, while in adult and taller trees (more than 2.5 m) the terminal bud of lateral branches or the same portion of the crown should be monitored. Attention should be also paid to the acropetal or basipetal sense of phenology in the crown. I.e., in wild cherry, phenology proceeds in an

30 D2.1 – Common protocols and reference standards for selected traits and species

acropetal way. That means that the crown should be ideally divided in 3 sectors to keep the same method in all the monitored trees (Figure 3.15.1).

Figure 3.15.1: For phenological surveys in adult trees, the crown should be ideally divided into 3 sectors.

The monitoring has to be performed in each sector and located in the same aspect. A phenological stage of a sector is reached when 50 % of buds show the characteristics describing a specific score. A sufficient number of trees to perform statistical analysis must be surveyed. When clones are considered, the number of plants per clone can be sensibly reduced to 3 – 5, while when progenies or provenances are monitored, the number can vary between 20 and 30 or more trees. Based on scientific questions and on funding resources, monitoring can be either conducted following a regular timing or just occasionally. The most common criteria are: - twice a week during all the flushing period. Each tree is monitored at each assessment; - at a given periodicity (days/week) during the flushing period by assessing only trees having reached a given score or all trees at different scores; - only once or twice during the flushing season when distances are an important limiting factor for surveys. The monitoring season should start soon enough to capture early stage and end late enough to catch the last stage. Each flushing assessment is to be carried out on the same day, or with a difference of one day maximum.

31 D2.1 – Common protocols and reference standards for selected traits and species

To reduce errors due to a subjective attribution of scores, the same staff should be involved in the survey. Monitoring can be carried out either in controlled or semi controlled environments (growth chamber, greenhouse) or in field (nursery, plantation). Anyway in field trials and plantations in general it is suggested that the monitoring start at least two years later in order to reduce the acclimation effect. The observation will stop when all trees have reached the last stage.

Scoring scheme The scoring system is the same for either in young and adult trees, but in the first case the assessment is carried out on the apical bud, while in the second one it is on the branch apical buds within a sector. A 5-step-scale is considered (Table 3.15.1; Figures 3.15.2 and 3.15.3).

Table 3.15.1. 5-step-scale used for wild cherry flushing monitoring Scale values Text 1 Buds not active, scales brown and closed (dormant winter bud) 2 Buds expanding, scales start to separate 3 Bud-burst (first green is visible) 4 Leaves are flushing 5 Leaves are fully expanded and new sprouts are in elongation

Figure 3.15.2: 5-step scale for young trees

32 D2.1 – Common protocols and reference standards for selected traits and species

Figure 3.15.3: 5-step scale for adult trees

For P. avium an integrated approach for phenology assessment can be also considered that combines bud break, flowering and ripening of fruits (DUCCI et al. 2012). Scores used can be compared with phenological growth stages defined by BBCH scale (MEIER 1997, MEIER et al. 2009) for stone fruit (Table 3.15.2 and Figure 3.15.4).

33 D2.1 – Common protocols and reference standards for selected traits and species

Table 3.15.2: BBCH Principal growth Stage 0 (sprouting/Bud development) and Stage 1 (leaf development) and BBCH-identification keys of stone fruit. BBCH growth stage Text 00 Dormancy: leaf buds and the thicker inflorescence buds closed and covered by dark brown scales 03 End of leaf bud swelling: scales separated, light green bud sections visible 10 First leaves separating: green scales slightly open, leaves emerging 11 First leaves unfolded, axis of developing shoot visible 19 First leaves fully expanded

Figure 3.15.4: BBCH score scale for stone fruit. Scores from 00 to 31will be considered in this framework.

This scoring system can be expensive in terms of time/personnel (a person is on average able to sample about 200 trees/day according to the size of trees) and it is suggested to be applied within collections, with special attention to reference clones.

34 D2.1 – Common protocols and reference standards for selected traits and species

References DUCCI F, DE CUYPER B, PÂQUES LE, PROIETTI R, WOLF H, (Compilers), 2012: Reference protocols for assessment of trait and reference genotypes to be used as standards in international research projects. Adaptive traits: Protocol nr. 8 – Definitions/recommendations; Protocol nr. 8.7 and Protocol nr. 8.12. Ed. CRA SEL - Arezzo, Italy: p. 82. DUCCI F, (ed), 2005: Monografia sul Ciliegio Selvatico (Prunus avium L.) [Monograph on wild cherry (Prunus avium L.)]. CRA – Istituto Sperimentale per la Selvicoltura, Arezzo, Italy, 126 p. ISBN 88- 901923-0-5 DUCCI F, SANTI F, 1998: The distribution of clones in managed and unmanaged populations of wild cherry (Prunus avium). Can J For Res 27: 1998–2004. DUCCI F, DE ROGATIS A, PROIETTI R, 2009: ‘Clonal replicated provenances’ performance and clone selection techniques. (Leopold Poljakovic-Pjnik and Albina Tarjan Tobolka Eds.) Proceedings of the international Conference “Forestry in achieving Millennium Goals”, Inst. of Lowland Forestry and Environment, Novi Sad: 93–103. MEIER U: BBCH (Stone fruits), http://www.bba.de/veroeff/bbch/bbcheng.pdf MEIER U, 1997: Growth stages of Mono- and Dicotyledonus Plants. BBCH Monograph, Blackwell Wissenschafts-Verlag Berlin Wien. MEIER U, BLEIHOLDER H, BUHR L, FELLER C, HACK H, HESS M, LANCASHIRE PD, SCHNOCK U, STAUSS R, VAN DEN BOOM T, WEBER E, ZWERGER P, 2009: The BBCH system to coding the phenological growth stages of plants – history and publications. J. Kulturpflanz 61: 41–52. NANSON A, 2004: Génétique et amelioration des arbres forestiers. Le Presses Agronomiques de Gembloux, A.S.B.L., Belgique: 712 p. (ISBN 2-87016-070-4).

1.151.2 Leaf senescence Definition Leaf senescence corresponds to the period when progressive changing of leaf color is observed. Leaf color can be characterized by high heritability, with special regard to clonal materials. Under local conditions relatively homogeneous color can be an important source of information for recording adaptive variation. Spatial micro-environmental variations, extreme environmental events (e.g., drought), the plant health conditions (bacterial/fungal spots), the sampled leaf amount and acropetal/basipetal senescence (thus the crown has to be divided into 3 sectors) represent sources of measurement error.

Description of the assessment in general For leaf senescence (yellowing) a subjective scoring system allows trees to be classified according to a grid representing different stages of tree yellowing. Observation is best done on small trees like in a nursery (but transplantation shock could be a source of error) or in field experiments (with trees of heights up to 3-4 m). Generally the whole crown of trees is observed.

35 D2.1 – Common protocols and reference standards for selected traits and species

Scoring scheme Assessment of senescence via a scoring system is generally used. It is a very subjective method. The organization of the survey is the same as for flushing. The senescence progress within the crown is acropetal, starting the first color variation from the bottom towards the tip. So, even in this case observations have to be divided among the three crown sectors or focused on only one of them. The BBCH system allows a subjective evaluation of color phases to be avoided and this system is again suggested. The BBCH growth Stage 9 (Senescence, beginning of dormancy) for stone fruit is reported in Table 3.15.3.

Table 3.15.3: Principal growth BBCH Stage 9: Senescence, beginning of dormancy. BBCH growth stage Text 91 Shoot growth completed; foliage still fully green 92 Leaves begin to discolor 93 Beginning of leaf fall 95 50% of leaves discolored or fallen 97 All leaves fallen 99 Harvested product (not to be considered in the framework of T4F)

A further possibility to evaluate senescence is offered by the use of colorimeters. These allow yellowing to be assessed via quantitative methods. At the clone level these traits can be highly heritable (0.67 on average for sensu lato heritability). Data can be managed as quantitative traits using a Portable Colorimeter Microflash v4.0. (methodology is described in wood color sheet).

3.152 Wood color

Definition Wood color is an aesthetic trait, for which special attention is placed in wild cherry and in broadleaves because wood is used to make valuable furniture and cabinetry. Then the evaluation of wood color is useful for both the characterization of reproductive materials and for the wood industry.

Assessment Quantitative parameters of the physical components of the color are evaluated using colorimeters, portable tools currently adopted by the paint or tissue industries for standardizing their production. Colorimeters using standard illuminants (mainly white light illuminants), and recording the reflexed light components, are able to measure basic parameters with very good approximation. Data can be managed as quantitative traits according to usual statistic methods.

36 D2.1 – Common protocols and reference standards for selected traits and species

Wood color, both of hardwood (duramen) and soapwood can be measured using: 1) destructive method, on circular sections of the trunk at different heights (at the base, at breast height and at 3-m); 2) non-destructive method , taking cores at the same heights from trees about 15-20 years of age (or > 20cm DBH).

Figure 3.15.5: Colorimeter Portable Microflash v4.0 and colorimetric coordinates

The Colorimeter Portable Microflash v4.0 (Figure 3.15.5) provides data relating to the colorimetric coordinates of the 3 system CIEL * a * b *: • L* - Luminance: is the variation of the color component along the black to white axis (z,); • a*: is the variation of the color component along the green to red axis (x); • b*: is the variation of the color component along the blue or yellow axis (y). Other parameters can be derived from the above mentioned ones, such as: white index (expressing the darkness/whiteness of the sample), C* (chroma/saturation; expressing the intensity of the color measured) and h (taint angle; expressing the height of the sample in the color space).

Methods

The color measurements (BOURGOIS et al. 1991) are carried out in the CIE- L*a*b* colour space using spectrophotometers with a standard illuminant D65 and 10° circular illumination. The color coordinates are calculated from the reflection data on the sample surface. In this color range, a colour is defined by its Cartesian chromatic coordinates: lightness L* (varies from 0 - black to 100 - white) and a* and b* (these coordinates define the chromatic plane; negative values of a* indicate green, while positive values indicate red; negative values of b* indicate blue, while positive values indicate yellow). The difference in chromaticity is defined as: ΔC = [(Δa*) 2+ (Δb*) 2]1/2.

The CIE L*a*b* system produces a fair correlation with the visual perceptions (CHRISMENT 2000). Concerning the color development during irradiation, it is useful to present the data in the CIE L*C*h* system which is more uniform (closer to the psycho-sensory assessment). The

37 D2.1 – Common protocols and reference standards for selected traits and species

color development can be derived simply from the CIE L*a*b* system by changing the Cartesian coordinates to cylindrical ones in the chromatic plane [a*, b*]. The Chroma (concentration or degree of color saturation) is defined then by C* = (a*2 +b*2)½ and the hue (saturation angle) h = arctan (b*/a*). The mean values of chromatic coordinates are calculated from measurements at different points on the radial surface of the sample (30x30 x10 mm, LxTxR). Tools/Equipment: quantitative or relative data can be recorded with spectrophotometers by different brands, i.e.,: - Spectrocolorimeter Datacolor ®Portable Microflash v4.0, with a measurement opening of 3 mm, - Konica Minolta spectrophotometer CM-2600 ®with D65 illuminant, a standard angle of 10° and a measurement opening of 8 mm.

References BOUGOIS J, JANIN G, GUYONNET R, 1991: La mesure de couleur: une méthode d'étude et d'optimisation des transformations chimiques du bois thermolysé. Holzforschung 45 (5): 377-382. CHRISMENT A, 2000: Color by the numbers. CD ROM Datacolor International, 3C-Conseil. DUCCI F, GERMANI A, JANIN G, PROIETTI R, SIGNORINI G, 2006: Clone selection for wild cherry (Prunus avium L.) with special reference to some traits used. In: BOZZANO M, RUSANEN M, ROTACH P, KOSKELA J, (compilers). EUFORGEN Noble Hardwoods Network, Report of the sixth (9-11 June 2002, Alter do Chão, Portugal) and seventh meetings (22-24 April 2004, Arezzo, Italy). International Plant Genetic Resources Institute, Rome, Italy: 53-60. FIORAVANTI M, SIGNORINI G, DUCCI F, 2009: Prunus avium L.: uno studio sull'ereditabilità del colore del legno. [Prunus avium L.: a study on the heritability of the wood color]. Act of the III Selvicolture National Congress, Taormina (ME), 2008 October, Vol. 3: 1498-1503. EL BAKALI I, YAGI S, MERLIN A, DEGLISE X, 2011: A Screening Study of Natural Colour of Wood from Different Geographical Regions. Research Journal of Forestry 5: 162-168. 10.3923/rjf.2011.162.168 JANIN G., 1987: Mesure de la couleur: Interet forestier et industriel. Ann. Sci. For., 44: 455-472. JANIN G, GONCALEZ J, ANANIAS R, CHARRIER B, DILEM A, DA SILVA F, DILEM A, 2001: Aesthetics appreciation of wood colour and patterns by colorimetry: Part 1. Colorimetry theory for the cielab system. Madears Ceincia Technol. 1: 3-11.

38 D2.1 – Common protocols and reference standards for selected traits and species

3.16 Broadleaves non-species specific protocols

Core group (broadleaves species leader): Mirko Liesebach (vT now THÜNENI; beech), Joukje Buiteveld (ALTERRA; common ash), Fulvio Ducci, Roberta Proietti (CRA; wild cherry), Marek Rzonca (IBL; oak), Marijke Steenackers (VLAGEW; poplar), Sven M.G. de Vries (ALTERRA; poplar)

The non-species specific traits for broadleaves describe the quality. These traits have an effect on the economic value of the final product. The final product is the trunk, particularly the lower part of the trunk.

3.161 Straightness

Definition A non-species specific trait for broadleaves is straightness. Stem straightness is under genetic control and can be affected by environmental conditions. Results from progeny trials show large differences in the frequency of straightness classes.

Description of the assessment in general Scoring straightness should be started when the trial has a mean height of 5 m. From then on straightness can be assessed without an upper height limit. The assessment has to be concentrated on about ¾ of the tree height beginning from the ground. That means the straightness in the crown is not taken into the assessment. The significance of the assessment increases with the age which is correlated with the height of the tress. In case of forked stems, only the trunk below the deepest forking point is evaluated. The assessment can be carried out during the whole year, particularly in older trials.

39 D2.1 – Common protocols and reference standards for selected traits and species

Scoring schema A 5-step-scale is recommended: Scale values Text 5 absolutely straight stem 4 fairly straight (in one direction slightly crooked) 3 slight to moderate bends in different directions 2 moderate to strong bends 1 no straight stem

5 4 3 2 1

Pros and cons of the described protocols a) Pros There is good experience in using the protocols. In several investigations a simplified version (only 3 steps were assessed: 1, 3, 5). b) Cons There is a slight risk that the straightness of a thinner tree will be classified better than those of a thicker tree.

References

KLEINSCHMIT J, SVOLBA J 1995: Intraspecific variation of growth and stem form in Quercus robur and Quercus petraea. In: Proceedings of the workshop, Brussels (BE), 15-16 June 1994. European Commision DG XII-E.2: 217-238

HOFMANN M, 2014: Hornbeam-accompanying tree species with perspective? Results of a provenance trial in Lower Saxony. Landbau forsch. Appl Agric Forestry Res 64: 99-106.

40 D2.1 – Common protocols and reference standards for selected traits and species

3.162 Forking

Definition A non-species specific trait for broadleaves is forking. Forking is can be caused both under genetic control and as damage to the terminal bud affected by environmental conditions. Forking can result by biotic and abiotic effects, for example frost events and deer browsing, respectively. Results from progeny trials show differences in the frequency of forking classes. Compared to a ramicorn, a fork has two leaders of equal importance in thickness and length, while a ramicorn is a branch thicker than mean branches, but thinner and usually shorter than the main stem.

Description of the assessment in general Presence versus absence of a fork is the most straightforward method. According to the height at which a fork appears, the effect can be judged detrimental or not. Scoring forking should be started when the trial has a mean height of 5 m. From then on forking can be assessed without any height limit.

Scoring schema For trees up to a mean height of 2 m of a trial series a 5-steps-scale is recommended: Scale values Text 5 No fork 4 Branch (ramicorn; thinner than a fork) with a similar angle 3 Fork(s) only in upper half of the tree height 2 Fork(s) only in the lower half of the tree height 1 Forks developed both in upper and in lower half of the tree height

½ h

5 4 3 2 1

41 D2.1 – Common protocols and reference standards for selected traits and species

Pros and cons of the described protocols When felling a tree with a fork, there is a risk that the trunk below the fork will tear. The parts above the forking point are thinner than the trunk below. This means the economic value of the trunks is smaller than a continuous thick trunk from the ground to the crown. An additional counting of the forks is therefore unnecessary. a) Pro There is experience in using the protocol to assess forking. b) Con It is sometimes difficult to distinguish between the reasons of forking, in particular when a tree has one fork. In the case of many forks in a tree, a genetic effect is presumable. A change from a fork to a ramicorn is fluent.

References

NW-FVA (not published) cited in PÂQUES L 2009: European survey on assessment methods of morphological traits. Treebreedex Doc. 916

3.163 Branch habit

3.163.1 Branch angle Definition Branch angle is important for the wood quality and has an effect on economic value. A steep branch is longer in wood then a plain growing branch. Between a steep growing branch and the stem, water can accumulate and can cause rotting.

Description of the assessment in general Branch angle is defined as the angle between a given branch and the main stem axis. The angle is measured at the insertion point. Practical assessment: dividing the angle between a rectangular growing branch and the stem into 3 angle classes with the same size. The scoring schema can be used at any age but preferable beyond the juvenile stage when the tree architecture stabilizes. For the assessment the mean branch angle class of all branches should be regarded.

42 D2.1 – Common protocols and reference standards for selected traits and species

Scoring schema

Scale values Text 1 <30° (steep) 2 30°-60° 3 60°->90° (plain)

1 (<30°) 2 (30°-60°)

3 (>60°)

Pro and con of the described protocols a) Pros The assessment can be carried out easily. b) Cons There is the danger of overestimation in the case of concentrating on a single branch, which is wrongly in focus. The reason for such a branch might be environmental.

3.163.2 Branch thickness Definition Branch thickness is important for the wood quality and has a direct effect on the economic value. A thin branch is better for the wood quality. The cutting point is restored soon after natural and artificial pruning.

43 D2.1 – Common protocols and reference standards for selected traits and species

Description of the assessment in general Direct measurement is very time consuming. Therefore a 3-step scoring is recommended. The trait branch thickness is provided as a relation of branch thickness to stem diameter. To get a feeling for branch thickness it could be helpful to measure a number of branches.

Scoring schema 3-step scale Scale values Text

1 Øbranch / Østem <= 0.3 (fine branches)

2 0.3 < Øbranch / Østem < 0.6 (moderate branches)

3 Øbranch / Østem >= 0.6 (thick branches)

Pros and cons of the described protocols a) Pros Having the feeling it is easy to assess. b) Cons Time is needed to get the feeling for evaluation. An underestimation of thicker trees can happen because thinner trees give the impression of having thinner branches.

44 D2.1 – Common protocols and reference standards for selected traits and species

3.2 Conifers

3.21 Norway spruce specific protocols

Core group: Luc Harvengt (FCBA), Jan Kowalczyk, Marek Rzońca (IBL), Egbert Beuker (METLA now LUKE), Mirko Liesebach (vTI now THÜNEN)

3.211 Flushing

Definition Bud break and bud set are among the most heritable traits of Norway spruce (Picea abies [L.] Karst.). Provenance tests show a great variability between populations in flushing. Provenances originating from higher elevations flush earlier than provenances from lower elevations. Also provenances originated in the northern part of the distribution area need a lower amount of temperature sum to flush and therefore, flush earlier than those originated in the south. Genotype's ranking for bud break is also very stable across sites and years.

Description of the assessment in general Bud break should be assessed separately for each tree in the field. Part of the crown to be assessed: Preferably the top of the crown (light crown) should be visible from one observation point. If this is not possible, then the middle part of the crown is also acceptable. The same part of the crown should be considered for subsequent phenological observations throughout the whole flushing period, as well as for subsequent years.

Scoring schema 5-step scale Scale values Text 1 Dormant winter bud 2 Starts flushing (first green is visible at the tip) 3 First needles visible (“brush stage”) 4 New shoot app. 3 cm 5 New shoot > 4 cm

45 D2.1 – Common protocols and reference standards for selected traits and species

1 2

3 4

(Photos: M. Liesebach, vTi) 5

Pros and cons of the described protocols a) Pros: The described protocol is very easy to carry out on a large number of trees per time unit. b) Cons: When observed once in a test, it is not always easy to get at the right time to have a good flushing differentiation between the genetic units.

References

SCHMIDT-VOGT H, 1987: Die Fichte. Band I [Norway spruce. Vol. 1]. P. Parey, Hamburg, Berlin: 647 pp.

46 D2.1 – Common protocols and reference standards for selected traits and species

3.22 Douglas-fir specific protocols

Core group: Jean-Charles Bastien (INRA), Daniel Michaud (FCBA), Jan Kowalczyk (IBL), Mirko Liesebach (vTI now THÜNEN)

3.221 Phenology

Definition Bud break and bud set are among the most heritable traits of Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). Provenance tests show a great variability between natural populations for bud flushing date. Interior provenances, which generally originate from high elevation, flush earlier than coastal provenances, whereas in the coastal part of the range, flushing date is mainly driven by the latitude of the geographic origin. Bud break and bud set are very important adaptive traits, related to the tolerance to abnormal climatic events that could occur in the growing environment. Very easy to score on a large number of (young) trees per time unit, these phenology traits are very useful to indirectly assess frost hardiness and, in some extent the stem form (e.g., forking). Genotype's ranking for bud break and bud set are also very stable across sites and years.

3.221.1 Flushing Description Flushing is recorded on each tree separately. The assessment method depends on whether the terminal bud is visible or not.

Scoring scheme When terminal bud is visible, a score is given to each tree according to the development stage of the terminal bud. Bud break on terminal bud (5-step score) Scale values Text 1 Dormant bud 2 Bud enlarges, slightly greenish appearance 3 Bud scales start to burst; needle points are visible 4 The needle bundle stretches up to twice the size of the bud size; in general the needles are still tight to the shoot; only rarely are needles around the bud scales splayed out 5 Needle bundle starts to loosen, shoot still enlarges; the young shoot looks like a paint brush

47 D2.1 – Common protocols and reference standards for selected traits and species

Stage 1 Stage 2 Stage 3

Stage 4 Stage 5

When the observation is made only once in the growing season, ideal time of recording is when around half of the trees have reached Stage 3. Flushing is better assessed when it is possible to record it repeatedly (e.g., beginning and end of flushing season during a given year or over two different years). When possible, an alternative (and more accurate) method consists to score 1 or 0 for each tree on a regular time basis (2 to 3 times a week), when the terminal bud reaches Stage 3. This method enables bud break to be expressed in days or degree-days (from a given origin).

When terminal bud is not visible (total height above 3m), a score is given to each tree according to the estimated percentage of crown buds having reached the development Stage 3 described above. Bud break on the whole tree (5-step score) Scale values Text 1 All buds dormant or just enlarged 2 1-25 % buds reach Stage 3 or more 3 26-50 % buds reach Stage 3 or more 4 51-75 % buds reach Stage 3 or more 5 76–100 % buds reach Stage 3 or more

48 D2.1 – Common protocols and reference standards for selected traits and species

Pros and cons of the described protocols a) Pros: The protocols described above are very easy to carry out on a large number of trees per time unit (several thousand per day). They enable a very efficient screening of genotypes for late frost resistance and, indirectly for lowering forking defects. b) Cons: For a given trial, bud flushing assessment has to be done in the shortest time possible (ideally within a day). When observed once in a test, it is not always easy to get the right time to have a good flushing differentiation between the genetic units.

References

ACEVEDO‐RODRIGUEZ R, VARGAS‐HERNANDEZ JJ, LOPEZ‐UPTON J, et al., 2006: Effect of geographic origin and nutrition on shoot phenology of Mexican Douglas‐Fir (Pseudotsuga sp.) seedlings. Agrociencia (Montecillo), 40 (1): 125‐137. ADAMS WT, BASTIEN JC, 1994: Genetics of second flushing in a French plantation of coastal Douglas‐fir. Silvae Genetica, 43 (5/6): 345‐352. BAILEY JD, HARRINGTON CA, 2006. Temperature regulation of bud‐burst phenology within and among years in a young Douglas fir (Pseudotsuga menziesii) plantation in western Washington, USA. Tree Physiology, 26 (4): 421‐430. CHEN Z, CLANCY KM, KOLB TE, 2003: Variation in budburst phenology of Douglas‐fir related to western spruce budworm (Lepidoptera: Tortricidae) fitness. Journal of Economic Entomology, 96 (2): 377‐ 387. LAVADINOVIC V, KOPRIVICA M, ISAJEV V, 2004: Phenological characters of Douglas‐fir provenances in Serbia. Silva Balcanica, 4 (1): 89‐94. OLSZYK D, WISE C, VAN ESS E, et al., 1998: Phenology and growth of shoots, needles, and buds of Douglas‐fir seedlings with elevated CO2 and (or) temperature. Canadian Journal of Botany, 76 (12): 1991‐2001.

3.221.2 Bud set Description Bud set assessment enables to indirectly evaluate hardiness to early fall frosts. This trait should be assessed on terminal bud (young trees) only.

49 D2.1 – Common protocols and reference standards for selected traits and species

Scoring schema Bud set on terminal bud (5-step score) Scale values Text 1 Terminal shoot is completely green, terminal bud is only allusively differentiated 2 Terminal bud is very small and visible between the terminal needles; terminal shoot is mainly green, sometimes slightly red 3 Terminal bud is conical and brown coloration starts; terminal shoot starts to lignify, cork cells are produced 4 Terminal bud is about 2‐3 mm, the brown color is more intense; bark appears mainly grey/greenish 5 Terminal bud is well developed and dark green; terminal shoot is lignified, that means the bark is greenish‐grey, and appear slightly teared

Pro and con of the described protocols a) Pros: The above described protocol is easy to carry out on a large number of trees per time unit (several thousand per day). When possible, it is suggested to assess bud set 2 or 3 times during the growth cessation period in order to better discriminate genotypes.

50 D2.1 – Common protocols and reference standards for selected traits and species

b) Cons: Bud set assessment requires a minimum training before starting evaluation on a large number of trees. This observation is generally more time consuming than flushing; therefore it is highly recommended to assess this trait at nursery stage rather than in the field.

References

PETKOVA K, RUETZ W, POPOV EMIL, et al., 2008: Testing of American, German and Bulgarian Douglas-fir progenies in experimental plantations in Bulgaria Austrian Journal of Forest Science 125 (2): 135- 156. ST CLAIR JB, HOWE GT, 2007: Genetic maladaptation of coastal Douglas-fir seedlings to future climates. Global Change Biology 13 (7): 1441-1454.

3.222 Frost Hardiness

Definition Frost damage is frequently observed in young plantations. In "frost-prone" sites, damage occurs more frequently than on milder sites and can be severe, leading to mortality, reduced growth and poor stem form. The greatest risk of cold injury occurs in the fall, before shoots are fully hardened for the winter or in the spring when shoots begin, or are about to begin, active growth. In the field, cold hardiness can be assessed after damaging frost events. However, in order to get a good statistical precision, it is important to ensure that incidence of natural frost injury is not sporadic across field test site and that frost damage can be separated from other causes of injury (e.g., drought, disease, insect). If not, artificial freezing tests on detached shoots should be preferred.

Description The following protocol is to be applied on a single tree basis. Subjective 5-step score: Scale values Text 1 Whole plant frozen to death 2 Terminal shoot frozen and dead, and lateral shoot moderately damaged 3 Severe damage on lateral shoots only 4 Little damage on lateral shoots only 5 No damage visible

51 D2.1 – Common protocols and reference standards for selected traits and species

Pros and cons of the described protocols a) Pros: The above protocol is very fast to implement on a large number of trees in nursery or in field tests. This evaluation is complementary to bud flushing or bud set assessment to screen genotype's cold tolerance. b) Cons: Frost hardiness assessment requires occurrence of a frost event (generally unpredictable) Frost Damage can sometimes be compounded with disease or insect damage.

References

BRAUN,H, 1998: Results of hybridization in Douglas fir (Pseudotsuga menziesii). Beiträge für die Forstwirtschaft, 22 (1): 1-7. HAWKINS BJ, STOEHR M, 2009: Growth, phenology, and cold hardiness of 32 Douglas-fir full-sib families. Canadian Journal of Forest Research 39 (10): 1821-1834. O'NEILL GA, ADAMS WT, AITKEN SN, 2001: Quantitative genetics of spring and fall cold hardiness in seedlings from two Oregon populations of coastal Douglas-fir. Forest Ecology and Management 149 (1/3): 305-318.

3.223 Needle cast

Definition Douglas-fir Swiss needle cast (Phaeocryptopus gaeumannii), is a foliar disease which originates from North America. It induces heavy needle losses, which generally start with the oldest ones. In case of severe attack, only last year's needles persist. This disease is favoured by a wet climate during the summer season or in wind-protected environments. Damages are observed mainly in young plantations (up to 15 years) and reveal most often mineral alimentation problems (weak growth, shallow soils, etc.). Although less heritable than growth traits, foliage traits appear to be reasonable indicator of Douglas-fir disease tolerance and can help screen for families that show tolerance to Swiss needle cast.

52 D2.1 – Common protocols and reference standards for selected traits and species

Description The following protocol is to be applied on a single tree basis. Subjective 5-step score: Scale values Text 1 Very severe infestation (76 % - 100 % needle loss) 4 Severe infestation (51 % - 75 % needle loss 3 Medium infestation (26 % - 50 % needle loss) 2 Very limited infestation (1 % - 25 % needle loss) 1 No infestation (0 % needle loss)

Pros and cons of the described protocols a) Pro: Quick and low cost evaluation method, which can be completed with an evaluation of foliage color (from 1= yellow o 3 = dark green) b) Con: This needle retention evaluation method does not enable to separate tolerance and resistance to Swiss needle cast.

References

HOOD IA, KIMBERLEY MO, 2005: Douglas fir provenance susceptibility to Swiss needle cast in New Zealand. Australasian Plant Pathology 34 (1): 57-62.

JOHNSON GR, 2002: Genetic variation in tolerance of Douglas fir to Swiss needle cast as assessed by symptom expression. Silvae Genetica 51 (2/3): 80-86.

TEMEL F, JOHNSON GR, ADAMS WT, 2005: Early genetic testing of coastal Douglas-fir for Swiss needle cast tolerance. Canadian Journal of Forest Research 35 (3): 521-529.

53 D2.1 – Common protocols and reference standards for selected traits and species

3.23 Scots pine specific protocol

Core group: Egbert Beuker (METLA now LUKE), Jan Kowalczyk (IBL), Volker Schneck, Mirko Liesebach (vTI now THÜNEN)

3.231 Lophodermium needle cast

Definition The needle cast disease caused by the fungus Lophodermium seditiosum is the most serious foliage disease of Scots pine (Pinus sylvestris) in the temperate zone. In some years heavy infections can cause severe defoliation to young pine stands over large areas and a reduction of tree growth. Therefore, the search for resistant or less susceptible populations or individuals is of great significance.

Description of the assessment in general The assessment of needle cast can easily be recorded at younger plants up to a height of 1 m (average height of a trial series). The whole plant has to be observed. The assessment has to be carried out in spring.

Scoring schema

Scale values Text 1 Very severe infection, all needles brown or dropped off 2 Medium to severe infection, 50-100 % brown needles 3 Medium infection, 10-50 % brown needles 4 Weak infection, few brown needles 5 No infection

54 D2.1 – Common protocols and reference standards for selected traits and species

1. Very severe infestation, all 2. Medium to severe 3. Medium infestation, 10-50 needles brown or dropped infestation, 50-100 % brown % brown needles off needles

Photos: S. ŚLUSARSKI - IBL

4. Weak infestation, few 5. No infection brown needles

Pro and con of the described protocols The scheme is to easy-to-use. Also the small size of the trees is an advantage for the assessment. Sometimes it can be difficult to distinguish the damage by Lophodermium seditiosum from other sources of browning of needles. For the statistical analysis it also has to be taken into account that the distances between the single values of the scale are not equal. So analysis of variance and estimation of variance components are not possible.

55 D2.1 – Common protocols and reference standards for selected traits and species

References STEPHAN BR, KRUSCHE D, 1986: Genetic variation of resistance to Lophodermium needle cast in Scots pine progenies of intraprovenance and interprovenance crossings. In Peterson GW (ed.): Recent research on conifer needle diseases. USDA, Forest Service, GTR WO-50: 28-34.

3.232 Pine twisting rust

Definition The pine twisting rust is caused by the fungus Melampsora pinitorqua. It causes severe damage to the shoots of Scots pine seedlings and young trees, resulting in growth reduction, malformation or even death, especially of seedlings. The pine twisting rust is very common in the northern temperate and boreal areas, where also European aspen (Populus tremula L.), which is the alternate host of the pathogen, is common. The pathogen enters the pine seedlings in spring and early summer during shoot elongation. The occurrence of pine twisting rust varies largely between years, due to its strong dependence upon various weather factors. There is large genetic variation in the resistance of Scots pine against the pine twisting rust, both within and between populations.

Description of the assessment in general Damage by the pine twisting rust can be distinguished most clearly at the end of the summer, when the effected parts of the shoots show resin secretion and become black. The assessments should be made at a young stage, up to a height of about 1 meter, assessing the whole tree. Due to large annual variation in the appearance of the disease, assessments should be made during years when the disease is common or, alternatively, repeated assessments should be made over several years.

Scoring schema Stem deformation by Melampsora pinitorqua: Scale values Text 0 present 1 lack

56 D2.1 – Common protocols and reference standards for selected traits and species

(Photos: ANTTI POUTTU, Luke)

Pro and con of the described protocols The scheme is easy to use. Also the small size of the trees is an advantage for the assessment. Especially when not assessed at the right time, the damage may be confused with other shoot deformations, such as those caused by insects.

References

ANDERSSON B, DANELL, O, 1997: In Pinus sylvestris resistance to pine twist rust associated with fitness costs or benefits? Evolution 51: 1808–1814.

MATTILA U, 2005. Probability models for pine twisting rust (Melampsora pinitorqua) damage in Scots pine (Pinus sylvestris) stands in Finland. Forest Pathology 35: 9–21.

QUENCEZ C, BASTIEN C, 2001: Genetic variation within and between populations of Pinus sylvestris l. (Scots pine) for susceptibility to Melampsora pinitorqua Rostr. (pine twist rust). Heredity 86: 36– 44.

57 D2.1 – Common protocols and reference standards for selected traits and species

3.24 Mediterranean pines specific protocol

Core group: Eduardo Notivol (INIA), Allain Bailly (FCBA), Fulvio Ducci (CRA)

Mediterranean pines are not special from the point of view (need) of protocols standardization. For all generic traits (according to a simple ontology mainly grouped in growth and biomass, phenology, pests and diseases resistance and reproduction), the assessing protocols should be the same that used in others pines or conifers. The differential focus for this group of species is not “how” but “for what”, and this is because the main purpose of these assessments is related to specific adaptation to the particular Mediterranean environment. When conservation and landscape protection is considered more interesting than wood production the interpretation of the results from measurements are different even when the protocols are the same. For forest trees, adaptation is important for any kind of environment but when species have to face up to global change, some environments are more susceptible than others to the consequences of that change. In this context there are two groups of traits related with resilience facing drought stress and perturbances, mostly fire.

3.241 Drought

For drought tolerance a good compilation of proxy assessment is included at the end of the document. Drought tolerance is not a trait and it is dependent not only on the plant but on the environment. In addition measuring drought tolerance is not straightforward. Drought tolerance for the same genotype varies with the soil characteristics, air temperature, water availability, light intensity and their regimes. The interpretation of results from measurements of mortality, soil water content, photosynthetic parameters, transpiration, CO2 concentration, water potential, water use efficiency assessed by means of isotopic discrimination, phytoregulators concentrations, terpene acids, or other by-products from metabolism, etc. can be used for this purpose but without a standardized protocol due to the different possibilities and framework of testing.

3.242 Flowering

Regarding reproduction, flowering and fructification assessments are not different for the species group. Protocols for cones and flower counting are not different for the Mediterranean pines compared with other species while other traits related to reproduction

58 D2.1 – Common protocols and reference standards for selected traits and species

like cone serotiny or masting can be highly relevant for Mediterranean pines. Unfortunately there are not agreed protocols for these traits (but see HERNANDEZ-SERRANO, 2014). Apart from multinomial calibrated scales (0 to 3; 0 to 5 similar to Scots pine or Norway spruce), a continuous variable can be obtained by visually counting strobili (male or female) in a fixed time (for example, 15 seconds), following protocols used in Californian oaks (KOENIG et al. 2009). It has been used with good results in several ongoing papers (HERNANDEZ et al., SANTOS DEL BLANCO et al., CLIMENT et al.). A manual click counter helps homogeneity among observers and observations.

3.243 Polycyclism

Polycyclism (the ability for a plant to produce several flushes in the same growing season) can be contemplated as vegetative or reproductive. In Aleppo pine (Pinus halepensis), but also occasionally in maritime pine (Pinus pinaster), multiple female cone cycles can be observed. Reproductive polycyclism can be recorded as presence / absence in each tree or rate of reproductive polycyclisms compared to a fixed number of reproductive shoots.

59 D2.1 – Common protocols and reference standards for selected traits and species

Quantitative way of assessment is made by means of counting the number of twigs and number of cycles for rates calculation (PARDOS 2003, GIRARD et al. 2011).

3.244 Bark thickness

Bark can be a paramount trait in protective strategies against forest fires. This trait (bark thickness) is recorded by means of bark calipers with no special requirements. Assessment of this trait is usually made by cross measurements in two directions (N-S and E-W).

References

CLIMENT JM, CHAMBEL MR, LÓPEZ R, MUTKE S, ALÍA R, GIL L, 2006: Population divergence for heteroblasty in the Canary Island pine (Pinus canariensis, Pinaceae). American Journal of Botany 93(6): 840–8.

CLIMENT JM, SAN-MARTÍN R, CHAMBEL MR, MUTKE S, 2011: Ontogenetic differentiation between Mediterranean and Eurasian pines (sect. Pinus) at the seedling stage. Trees - Structure and Function (25): 175–186.

CLIMENT J, MARTÍN-SANZ R, SANTOS DEL BLANCO L , CHAMBEL MR, NOTIVOL E, 2014: Conference Paper: Genetic, environmental and ontogenetic effects on cone serotiny in Aleppo pine (Pinus halepensis Mill.) MEDPINE5. 5th International Conference on Mediterranean Pines; 09/2014

CLIMENT JM, et al., (in prep): Population differentiatidon for life history traits in Aleppo pine (Pinus halepensis Mill.)

CLIMENT JM, et al. (in prep): Divergence between subspecies of European black pine (Pinus nigra Arnold) on life history traits.

ESPELTA JM, ARNAN, X, RODRIGO A, 2011: Non-fire induced seed release in a weakly serotinous pine: climatic factors, maintenance costs or both? Oikos 120 (11): 1752–1760.

60 D2.1 – Common protocols and reference standards for selected traits and species

GIL L, LÓPEZ R, GARCÍA-MATEOS Á, GONZÁLEZ-DONCEL I, 2009: Seed provenance and fire-related reproductive traits of Pinus pinaster in central Spain. International Journal of Wildland Fire 18 (8): 1003.

GIRARD F, VENNETIER M, OUARMIM S, CARAGLIO Y, MISSON L, 2011: Polycyclism, a fundamental tree growth process, decline with recent climate change: the example of Pinus halepensis Mill. in Mediterranean France. Trees-Structure and Function, DOI: 10.10: 1–12.

HERNÁNDEZ, et al. (in prep.): Genetic structure of cone serotiny in Aleppo pine (Pinus halepensis Mill.)

KOENIG WD, KNOPS JMH, CARMEN WJ, SAGE RD, 2009: No trade-off between seed size and number in the valley oak Quercus lobata. The American Naturalist 173 (5): 682–8.

MAGINI E, 1969: The heritability of the stem form in Pinus pinaster Ait. 2ème Consultation Mondiale sur l’Amélioration des arbres forestiers. Washington FO-FTB 3/8: 351-360.

MARTÍN-SANZ R, NOTIVOL E, SANTOS-DEL-BLANCO L, SAN MARTÍN R, CHAMBEL MR, CLIMENT J . Phenotypic plasticity and allometric effects of cone serotiny in a Mediterranean conifer (In prep.)

PARDOS M, CLIMENT J, GIL L, PARDOS JA, 2003: Shoot growth components and flowering phenology in grafted Pinus halepensis Mill. Trees Struct Funct 17: 442-450

SANTOS DEL BLANCO et al., (in prep.): Quantitative genetics of male and female reproduction in pines.

61 D2.1 – Common protocols and reference standards for selected traits and species

3.25 Larch specific protocol

Core group: Luc Pâques (INRA), Jan Kowalczyk (IBL), Ecaterina Nicoleta Chesnoiu (ICAS), Volker Schneck (vTI now THÜNEN)

3.251 Phenology

Definition Bud burst and bud flushing are the most critical parts of phenology in larch as they are directly related to the risk of late frost damage. They are also a clear signal of re-start of growth of primary meristems. A large variability at population and individual levels is observed for that trait.

Way of assessment recommended

For young trees, that is up to a height the terminal bud is easily observable (2-3 m), observation of bud flushing can be limited to the terminal bud. In this case, at each date of observation, a score is given following the scoring-scale. Observation can be done weekly if the kinetics of flushing is of interest; if not, it can be done at only one date when 50 % of the trees are at Stage 3.

62 D2.1 – Common protocols and reference standards for selected traits and species

Score

For older and taller trees, that is when total height prevents an easy observation of the terminal bud (above 3 m), the international BBCH scoring system will be favoured and has been adapted to larch. The observation considers all vegetative buds of the whole crown. Scores range from 0 up to 31. Scores 0 to 10 consider the phase up to the first buds reached score 5; scores 11 to 19 evaluate the proportion (from 10 % to 90 %) of buds having reached stage 5; score 31 corresponds to the stage when bud flushed is achieved and first twig elongation is observed. As for young trees, observation can be done weekly or at only one date. Trees are considered flushed at the date when they reached BBCH-score 15.

63 D2.1 – Common protocols and reference standards for selected traits and species

Code Stage Description BBCH 0 leafing All vegetative buds at stage 0 (dormant) 1 leafing Most of buds have reached stage 1 3 leafing

7 leafing Most of buds have reached stage 2 9 leafing Most of buds have reached stage 3-4 10 leafing First buds have reached stage 5

11 leafing 10% of buds with score 5

12 leafing 20% of buds with score 5

13 leafing 30% of buds with score 5

14 leafing 40% of buds with score 5

15 leafing 50% of buds with score 5

16 leafing 60% of buds with score 5

17 leafing 70% of buds with score 5

18 leafing 80% of buds with score 5

19 leafing 90% of buds with score 5

31 growth Start of twigs elongation

3.252 Canker

Definition Larch canker due to Lachnellulla willkommii is the most common disease observed in European larch, especially in alpine populations, but Sudetan and Central Poland populations are not exempt. Canker produces deformation observable on branches and on stem, including at some stages, resin flow and presence of fructification. In the worse cases, girdling of the stem might be complete.

64 D2.1 – Common protocols and reference standards for selected traits and species

Method of assessment Observations can usually start when trees are around 10 years old, rarely before except in case of artificial inoculation. - Artificial inoculation : Inoculation is done through insertion of a calibrated sorgho straw infected by Lachnellulla willkommii, in a hole drilled by a machine. A nearby hole is performed for control. Young trees (2 years old) and young organs (1-2 years-old branches) can be used (Sylvestre- Guinot and Delatour, 1983). Assessment is usually done 6 months after inoculation by measurement of the length (mm) of the canker, the appreciation of the proportion of the twig girdled (ex. 1/3) and by the notation of resin flow and of fructification (presence/absence).

- Observation in nature The severity of the disease is appreciated through the presence (1) vs absence (0) of symptoms (0) on stem or branches. The frequency of trees of a given genotype presenting canker is then calculated. (SCHOBER 1985).

Scoring schema

Scale values Text 1 Disease present on stem or branches 0 Disease absence

Method recommended: Artificial inoculation has the main advantage to allow a much earlier screening of trees and in a relatively fast way (6 months). Compared to observation in natural conditions, the inoculum source is controlled but limited, applied at a high concentration and through man- made damage (hole), which might amplify the response.

65 D2.1 – Common protocols and reference standards for selected traits and species

Reference

SYLVESTRE-GUINOT G, DELATOUR C, 1983: Possibilités d’appréciation de la sensibilité du genre Larix au Lachnellulla willkommii (Hartig) Dennis par inoculation artificielle. Ann.Sci.For. 40: 337-354.

SCHOBER R, 1985: Neue Ergebnisse des II.Internationalen Lärchenprovenienzversuches von 1958/59 nach Aufnahmen von Teilversuchen in 11 europäischen Ländern und den U.S.A. Schriften aus der Forstlichen Fakultät der Univ. Göttingen, Band 83.

3.253 “Drought” cracks

Definition Cracks are sometimes visible along larch stem. Their origin is not well known (growth speed, drought?) but is more frequent in drier sites (shallow soil, southern aspect). The defect may be considerable as the cracks usually turn around the stem over one to several meters and can be as deep as the pith. After a few years, the crack might be filled up through healing, which makes the defect less easily observable. But an attentive observation allows it to be detecte through the resulting cicatrix. All species of larch are susceptible, but more frequently this defect is observed on European larch than on hybrid and finally on Japanese larch.

Way of assessment recommended The observation requires a close look at stem and to turn around it to have a full vision as the cracks are not necessarily oriented and are usually hidden by branches. The best season is during winter time when needles have fully fallen off. The defect seems to appear at a juvenile stage when trees are 10-15 cm diameter thick at BH, which seems a critical stage.

66 D2.1 – Common protocols and reference standards for selected traits and species

Score A simple scoring system, namely presence/absence is used.

References

CAZAUX JP, CHEVALIER R, GILBERT JM, GINISTY C, 1994: Le mélèze hybride en plantation - résultats provisoires sur 17 sites en France. (Hybrid larch plantations - provisional results from 17 sites in France). Etudes du CEMAGREF: Série Gestion des Territoires 12: 149-162.

67 D2.1 – Common protocols and reference standards for selected traits and species

3.26 Conifer non-species specific protocol

Core group (conifer core group leaders): Luc Pâques (INRA; Larch), Luc Harvengt (FCBA; Norway spruce), Jean-Charles Bastien (INRA; Douglas-fir), Egbert Beuker (METLA now LUKE; Scots pine), Eduardo Notivol (INIA; Mediterranean pines)

3.261 Stem straightness

Definition Stem deformation includes several defects: basal sweep, lining, bending, crookedness, twisting. Their origin can be genetic (due to delayed lignification, production of compression wood?) or accidental. The trunk aspect may change over time with a more or less rapid apparent correction due to the accumulation of growth layers.

It is recommended to separately evaluate these different defects for a better description of stem form and a separation of accidental from genetic effects. Stem sinuosity or stem crookedness (vs Stem straightness) aim to assess the external quality of the stem in connection to local deviations from the main tree axis, present along the stem, in any direction. It also includes twisting defects common in some species (larch). Severity of deviations is more or less important: in some cases, the defects disappear externally after a few years, but in the worse cases, they remain over years and are highly detrimental to wood quality. These should be distinguished stem deformation due to intrinsic causes (like due to lignification problems) and stem defects clearly resulting from an

68 D2.1 – Common protocols and reference standards for selected traits and species

identified biotic (e.g., Rhyacionia buoliana in pines) or abiotic (such as frost/drought damage to terminal buds) factors. In the latter case, a rupture of direction at a given annual increment is observed. In larch species, stem crookedness is clearly a major defect which might be accidental but which has overall a strong genetic control.

Description of the assessment Age: For most studies, evaluation should be done when trees are enough developed and better express defects, that is when trees are between (5)-10-15-(20) years old. Afterwards, stem correction through diameter enlargement hides defects which nevertheless remain within the log. Season: Observation should be done during the winter time, when the stem is fully visible, that is in absence of needles. Assessment: it is recommended: 1) to note separately the different defects such as basal sweep, lining, bending and stem sinuosity, 2) to use the scoring scale in an absolute manner (agreed by participants in Leuven) which means that a given score must have the same meaning across different sites; 3) to limit thus observation of crookedness to the part of the stem above 1.30 m (to avoid basal sweep) and avoid the last 2 annual increments: in some species, this part of the stem is heavily influenced by local weather conditions (wind) and corrections of the stem appear in next season(s).

Before starting observation: 4) to walk through the plantation and find reference trees for each score, these trees will be marked and referred to several times if needed during the assessment. Picture of these trees should be taken and archived for further documentation.

During the observation: 5) to observe trees in two perpendicular planes, so to have a better vision of defects, 6) a given block will be assessed by only one observer, so that observer effects will be included in block effects, 7) in case of doubt between two scores, use the smaller one.

According to available resources, it is recommended to have notation by two independent observers.

69 D2.1 – Common protocols and reference standards for selected traits and species

Scoring scheme The following scoring system is proposed: it combines a count of crooks along the stem together with an appreciation of the severity of the crooks.

Scores 5 and 4 include what can be seen as ‘straight’ trees: defects in Score 4 are weak enough to suppose that they will disappear with time and have no or little effect on wood quality; Scores 1 and 2 include ‘crooked’ trees: the defect is so severe that it is supposed to remain over time and strongly affect wood quality (purge needed).

This scoring system can be used in two different ways: 1) in an “absolute” manner, which means that a given score will have the same meaning across different sites; 2) in a “relative” manner: which means to adapt the scoring scale to each site.

Pros and cons The advantage of the “absolute” manner is that it allows a real comparison of results over sites (and ages); the inconvenience being that in a given site, the distribution of scores will be asymmetric. The advantage of the “relative” manner is that it allows normalising the distribution of scores; but it prevents an objective comparison among sites. Comparison could only be done through genotype ranking with a loss of information. At the Leuven workshop (September 2009), participants agreed that the most important factor in international trial evaluation was the possibility to compare genotypes’ behaviour across sites and the absolute way was retained.

70 D2.1 – Common protocols and reference standards for selected traits and species

References KEIDING H., 1966: Aim and prospects of taek breeding in Thailand. Nat. His. Bull. Siam. Soc. 21 (1 & 2)

3.262 Basal sweep

Definition Basal sweep is a trunk deformation observed at the base of the tree. It can be of genetic origin or accidental (in connection with a poor plantation, poor weed control or wind damage, snow creepage on steep slope). Some species, like maritime pine, larch, etc., are highly susceptible to this defect

Assessment A subjective scoring (1/0: presence /absence of the defect) might be enough in most cases. When a more objective evaluation is needed, we recommend the following procedure:

1.30 m

The longer horizontal distance (d) between the deformed tree and a vertical line up to 1.3 m will be measured to evaluate the basal sweep. The vertical line is materialised by a pole standing at the base of the stump and maintained vertical (plumb line, or ). The measure (d) is expressed in centimeters.

Recommendation The deformation is observed in the plan where the defect is the most severe and up to 1.3 m.

Pros and cons Several other methods exist as shown in next figure.

71 D2.1 – Common protocols and reference standards for selected traits and species

Method 1 Method 2 Method 3 Advantage Most rapid & simple Adapted to all forms of Apparently convenient basal sweep and more method precise Inconvenient But not always 2 points Need a pole with a - needs 2 measures (150 cm of contact with trunk spirit level and a and HT), mobile gauge to - difficulties if weed at the measure deviation base of the tree - difficulties to measure HT with enough precision

Reference ème MAGINI E; 1969: The heritability of the stem form in Pinus pinaster Ait. 2 consultation mondiale sur l’amélioration des arbres forestiers. Washington FO-FTB 3/8. 351-360.

3.263 Forking

Definition Forking is considered when the main stem axis splits (usually in conifers) into 2 stems of equal importance (similar diameter). Other types of split of the main axis can occur when, for example, a vigorous branch takes an up-right position: in this case, the second axis is usually of a smaller diameter: it is then called ‘ramicorn’. Forking can be genetically inherited and/or be accidental due to wind, snow, frost, birds, etc., damages. Re-iteration of the defect is possible.

Assessment It is recommended to adopt different protocols according to the age/size of trees: For young trees (< 15 m), scoring simply identifies trees with or without a fork (1/0). For older trees (> 15 m), the assessment combines the position of forking occurrence and the number of forks:

72 D2.1 – Common protocols and reference standards for selected traits and species

No fork Fork Position: relative to tree Upper third Middle third Lower third height

Score relative to position 4 3 2 1

Number of axis (forks) 1 n1 n2 n3

Forking index = (score x 10)/nber of axis

Example: number of axes 1 2 2 2 Forking index 40 15 10 5

Recommendation For species with usually no forking re-iteration, the score relative to forking position may be suitable. For species with usual forking re-iteration, it is recommended to combine both records (position + number) into a forking index: the highest index being for non-forking trees and for trees with a lower number of forks at a higher position.

3.264 Branching habit

Definition Branching is commonly evaluated through 3 main parameters including branch thickness, branch angle and branch number of primary branches along the trunk. A high density of branches, coarse branches, and branches with an acute angle towards the trunk are indeed considered to downgrade overall stem quality and affect internal wood properties (due to

73 D2.1 – Common protocols and reference standards for selected traits and species

knots size, angle, density). Thin, flat angle branches at a low density are commonly searched for by breeders.

3.264.1 Branch angle A 4-scale subjective scoring system is used judging the angle made by branches to the main stem: the flatter the angle (close to 90° to the trunk), the better the branching.

Scale values Text 4 branch in the 1st quarter (angle between 67° and 90°angle close to 90°) 3 branch angle in the 2nd quarter (between 45° and 67°) 2 branch angle in the 3rd quarter (between 23° and 45°) 1 branch angle in the 4th quarter (below 23°)

The rationale of this scoring system is that an observer can quite easily figure out the 4 quarters of the angle between the trunk and a perpendicular horizontal line; firstly by dividing the 90° into two parts and then again into two parts. So the observer starts to position the branch in the first (45-90°) or in the second half (0-45°), and then respectively by dividing it in two, in the 1st or 2nd quarter or in the 3rd or 4th quarter.

Recommendation: Assessment is done at the age of 10 years after plantation, provided total tree height exceeds 6 m. The height (or whorl) at which branching angle is to be assessed will be decided within each trial network.

74 D2.1 – Common protocols and reference standards for selected traits and species

For some species, at certain ages and sites, branches can hang down at more than 90°. An additional score (5) could be added to take this pattern into consideration. For some species, branches can start from the trunk at a certain angle and then flatten onwards. Angle from the closest part of the branch to the trunk should be then considered.

3.264.2 Branch thickness A 4-scale subjective scoring system is recommended: it is made more objective by relating primary branch sizes (diameter) to stem diameter.

Step Description 4 branch diameter less than ¼ stem diameter 3 branch diameter between 1/3 and ¼ stem diameter 2 branch diameter between 1/2 and 1/3 stem diameter 1 branch diameter above ½ stem diameter

Recommendation Assessment is done at the age of 10 years after planting, provided total tree height exceeds 8 m. In any case, the branches should be easily visible from beneath the trees. The position of the whorl where branch thickness will be assessed (or the height of branches) will be decided within each trial network.

3.264.3 Branch number The total number of primary branches (above a certain diameter) is an important feature of branching but its consequences on stem and wood quality are also influenced by the way branches are distributed along the trunk. A two-fold scoring system is suggested combining number and distribution of branches:

75 D2.1 – Common protocols and reference standards for selected traits and species

Score 1: distribution of branches along the stem 1 = not in whorls 2 = in whorls Score 2: number of branches along the stem or in whorls 1 = many branches 2 = moderate number of branches 3 = few branches

Position Number of branches Not in whorls In whorls 1 2 Many 1

Moderate 2

Few 3

Comments - For some species like larch, branches are not organised in neat whorls so that the distribution score may be useless. In such cases, only the score for branch number will be used. - If operators are able to identify which branching distribution is more favourable (in relation to wood quality, …), one could consider combining scores so that the ‘best’ position and low branches numbers give the highest score (i.e., Low number in whorls = 3 x 2 = 6).

Recommendation - The height (or whorl) at which branching number is to be assessed will be decided within each trial network. - It might be recommended to fix the minimum diameter above which branches are counted.

76 D2.1 – Common protocols and reference standards for selected traits and species

4. Trait-oriented common protocols

4.1 Wood quality

Manuel Touza Vazquez (CIDTG) (comp.)

There is an increasing interest regarding how to determine wood properties in standing trees with non-destructive techniques (NDTs). This is of particular interest to breeders as these new possibilities may be introduced into breeding programs, allowing, at the same time, the preservation of the original trees. One of the best known examples is the recent application of acoustic methods for estimating the mechanical properties (modulus of elasticity) in standing trees. Other available technologies include penetrometers (pylodin), drilling resistance methods (resistograph), measurement of longitudinal surface growth strains with strain gauge devices (as the one developed by CIRAD-Forêt), etc. In other cases, traditional lab techniques together with X-Ray densitometry, fractometers, etc., allow the determination of an increasing number of wood properties (density, sapwood-heartwood ratio, early-wood-late-wood ratio, bending strength, compression strength…) in increment cores. NIR techniques also allow the determination of an increasing number of wood properties from wood cores and/or wood power. Due to the previous reasons, a questionnaire was drawn up on non-destructive methods for assessing wood properties in standing trees and distributed by CIDTG in September 2014 to WP2 and WP11 partners. The questionnaire was based on the outcome of a previous survey carried out in the frame of WP11 and was structured into four parts (General overview of available techniques and equipment; Acoustic tools; Near infrared Spectroscopy and Innovation, and future trends). The objective of the survey was to gain information on existing equipment and protocols for determining wood properties in standing trees as well as to identify gaps and future trends. For the purpose of the questionnaire, only wood properties determined on standing trees or small wood samples (as a wood core or wood power) may be considered as non-destructive as they will allow the preservation of the individual tree. Twelve partners from ten countries responded to the WP2 survey (Partners 1-INRA [FR], 3- ALTERRA [NL], 6-BOKU [AT], 8-CIDGT [PO], 9-CNR [], 10-CRA [], 12-FCBA [FR], 18-IICT [], 20- INNVENTIA [], 24-METLA (now LUKE) [FI], 26-UGent [BE] and 28-vTI (now THÜNEN) [DE]). The survey was structured into four parts: Part I. General overview of available techniques and equipment Part II. Acoustic tools Part III. NIR Part IV. Innovation and future trends

77 D2.1 – Common protocols and reference standards for selected traits and species

The responses were analysed in October 2014 and presented at the 3rd Annual meeting in Tulln (AT). The main results are discussed below.

4.11 General overview of available techniques and equipment

Amap was created of the available techniques that are employed and/or being evaluated for determining wood properties in standing trees. At the same time, several institutions are evaluating the future use of the same techniques.

Project partners from ten countries responded to the WP2 survey CNR INN- METLA BOKU CIDGT CRA FCBA IICT INRA WUR vTI* U Gent IVALSA VENTia (LUKE)

Pylodin

Resistograph Acoustic methods Wood cores Trad. Lab. Wood cores X-Ray densit. Wood cores NIR Wood cores Colorimeter Rigidimeter for MoE *now THÜNEN Codes employed: Employing the technique Evaluating the future use Most experience in other fields (timber construction)

Only four technique, comprise the 80 % of the total that are being employed for determining wood properties in standing trees.

78 D2.1 – Common protocols and reference standards for selected traits and species

Wood cores / Traditional lab. Procedures Wood cores / X-Ray densitometry Pylodin Wood cores and NIR Resistograph Acoustic methods Wood cores / Colorimeter Rigidimeter for MoE

Wood density in standing trees is mainly determined employing a wood core. Traditional volumetric methods and X-Ray densitometry are being employed in similar percentages.

Wood core and volumetric method Wood core and X-Ray densitometry Penetrometer (pylodin) Wood core and NIR

The next graph points out how the different techniques agree with the expectations of the users (1 Absolutely agree, 0,6 Rather agree, 0,3 Rather disagree, 0 Absolutely disagree).

Wood cores / Traditional laboratory procedures

Pylodin

Resistograph

Wood cores / X-Ray densitometry

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

79 D2.1 – Common protocols and reference standards for selected traits and species

Half of the institutions that answered the survey are evaluating new techniques for determining wood properties in standing trees. NIR, resistograph and acoustic methods comprise 80% of the techniques being evaluated

3.5

3

2.5

2

1.5

1

Number institutionsof 0.5

0 Resistograph Wood cores and Acoustic methods Wood cores / X- Pylodin NIR Ray densitometry

4.12 Acoustic tools

A map of the equipment being used, as well as the species tested.

Equipment CIDGT FCBA INNVENTIA Director ST 300 x x Microsecond Timer x IML Hammer x Hitman (x) Ultrasonic Timer x

Species tested CIDGT FCBA INNVENTIA Pinus pinaster x x Picea abies x Pinus taeda x Pseudotsuga menziesii x Eucalyptus spp (x) Populus spp (x)

80 D2.1 – Common protocols and reference standards for selected traits and species

4.13 Near Infrared Spectroscopy (NIR)

Six of the twelve partners that answered the questionnaire are using NIR and two others are planning to do in the future.

CNR METLA BOKU FCBA IICT INNVENTIA INRA U Gent IVALSA (LUKE) More than ten years x x x x Between 5-10 years x x Planning to do so in the x x future

Together with traditional properties such as chemical composition or density, NIR is being used increasingly in new applications such as wood identification, durability, surface weathering, etc.

6

5

4

3

2

Number institutionsof 1

0 Chemical Mechanical Physical Wood Durability Surface composition properties properties identification weathering

The partners were asked to select whether or not the following statements hold true for them (1 Absolutely agree, 0.66 Rather agree, 0.33 Rather disagree, 0 Absolutely disagree).

NIR may save money and time in the determination of wood properties 0.94 It is better to work with wood cores for having an idea of the radial variation of the property 0.94 NIR will have an increasing use in the forestry sector 0.83 It is better to work in the laboratory with a properly seasoned sample than try to determine a 0.78 quick reference value in the field NIR seem the most promising technique for determining new wood properties in standing trees 0.66 NIR equipment is expensive and require a well-trained, staff so its use will be restricted to a 0.61 reduced number of research institutions

81 D2.1 – Common protocols and reference standards for selected traits and species

The answers pointed out a clear agreement that NIR may save money and time in the determination of wood properties, and as well that it is better to work with wood cores to obtain an idea of the radial variation of the property.

4.14 Innovation and future trends

The next graph points out the main interests in employing NDTs in standing trees (1 Absolutely agree, 0.6 Rather agree, 0.3 Rather disagree, 0 Absolutely disagree). All the partners absolutely agree that their main interest in using NTDs it a better understanding of the effect of sylviculture on wood properties as well as to improving the traditional breeding programs introducing new criteria.

Look for the genetic gain associated with individual trees

Look for applications of the timber

Look for an early selection of the best provenances/clones

A better understanding of the effect of silviculture on wood properties

Improve the traditional breeding programs introducing new criteria

0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05

The next graph shows the future trends regarding the use of NDTs in standing trees (1 Absolutely agree, 0.6 Rather agree, 0.3 Rather disagree, 0 Absolutely disagree). There is a clear agreement that cooperation and the definition of common protocols are essential for the development of NTD´s.

82 D2.1 – Common protocols and reference standards for selected traits and species

In 10 years my organization will determine additional properties in standing trees

The information obtained from a wood core may give an overall idea of the whole tree The number of wood properties that may be determined with NDTs in standing trees it is still very reduced In 10 years, new properties will be determined with NDTs in standing trees

Cooperation and definition of common protocols, it would be essential for the development of NDTs

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

The following is a list of innovative methods that have been recently developed by the partners for the assessment of wood properties in standing trees: • CIDGT: Tension wood in standing Eucalyptus globulus • CIDGT: Acoustic methods for determining MoE in young trees (work in progress) • CNR-IVALSA: X-ray tomography (density distribution and 3D maps) • CNR IVALSA: Hyperspectral imaging (variation of wood properties in space, spectral maps) • IICT: Physical and chemical characterization using core sampling • INNVENTIA: Radial variations in spiral grain for some hardwood species (work in progress with larch) • INRA: Rigidimeter on larch and Douglas fir • U GENT: Microdensitometrical analysis of long wood cores with micro-CT

The partners were asked if they were interested in testing any particular wood property that cannot be predicted nowadays. The following table shows the answers.

CNR METLA vTI BOKU FCBA IICT INNVENTIA INRA IVALSA (LUKE) (THÜNEN) Vulnerability to drought stress x Complete characterization of material – x multisensory approach Retrospective assessment of annual x growth Hydraulic conductivity using small x branches in Pinus pinaster Whole-timber wood homogeneity and x performance during wood drying Chemical composition and heat value x Spiral grain, Heartwood/sapwood limits x NIR models for the constitutive stilbene content in Scots pine heartwood or x induced content in other tissues

83 D2.1 – Common protocols and reference standards for selected traits and species

References ALVES A, SANTOS A, ROZENBERG P, PÂQUES L, CHARPENTIER JP, SCHWANNINGER M, RODRIGUES J, 2012: A common NIR-based PLS-R model for the prediction of basic wood density of Pinus pinaster and Larix × eurolepis. Wood Science and Technology 46: 157–175 (DOI: 10.1007/s00226-010-0383-x). ALVES A, SANTOS A, SILVA-PEREZ D, RODRIGUES J, PEREIRA H, SIMÕES R, SCHWANNINGER M, 2007: NIR PLSR model selection for Kappa number prediction of Maritime pine Kraft pulps. Wood Science and Technology 41: 491–499. ALVES A, SCHWANNINGER M, PEREIRA H, RODRIGUES J, 2006: Calibration of NIR to assess lignin composition (H/G ratio) in Maritime pine wood using analytical pyrolysis as the reference method. Holzforschung 60: 29-31. ALVES A, SCHWANNINGER M, PEREIRA H, RODRIGUES J, 2006: Analytical pyrolysis as a direct method to determine the lignin content in wood. Part 1: Comparison of Py-lignin with the Klason lignin method. Journal of Analytical and Applied Pyrolysis 76: 209-213. ALVES A, SIMÕES R, SANTOS C, POTTS B, RODRIGUES J, SCHWANNINGER M, 2012:Determination of Eucalyptus globulus wood extractives content by NIR-based PLS-R models: Comparison between extraction procedures. Journal of Near Infrared Spectroscopy 20: 275-285. ALVES A, SIMÕES R, STACKPOLE D, VAILLANCOURT R, POTTS B, SCHWANNINGER M, RODRIGUES J, 2011: Determination of the syringyl/guaiacyl (S/G) ratio of Eucalyptus globulus Labill. wood lignin by NIR-based PLS-R models using analytical pyrolysis as the reference method. Journal of Near Infrared Spectroscopy 19: 343-348. ARCINIEGAS A, PRIETO F, BRANCHERIAY L, LASAYGUES P. 2014: Literature review of acoustic and ultrasonic tomography in standing trees. Trees - Structure and Function 28, 6: 1559-1567. AUTY D, ACHIM, A. 2008: The relationship between standing tree acoustic assessment and timber quality in Scots pine and the practical implications for assessing timber quality from naturally regenerated stands. Forestry 81(4), 475-487.. BOUFFIER L, CHARLOT C, RAFFIN A, ROZENBERG P, KREMER A, 2007: Can wood density be efficiently selected at early stage in maritime pine (Pinus pinaster Ait.)? Ann. For. Sci. 65: 106-114. COWN D.J. 1982: Use of the Pilodyn wood tester for estimating wood density in standing trees - Influence of site and tree age. New Zealand Forestry Service, Forest Research Bulletin No. 13, Rotorua, New Zealand. DA SILVA PEREZ D, GUILLEMAIN A, ALAZARD P, PLOMION C, ROZENBERG P, RODRIGUES JC, ALVES A, CHANTRE G, (2007: Improvement of Pinus pinaster ait elite trees selection by combining near infrared spectroscopy and genetic tools. Holzforschung 61: 611-622. DOWNES G, TOUZA M.C, HARWOOD C, WENTZEL-VIETHEER M. 2014: NIR detection of non- recoverable collapse in sawn boards of Eucalyptus globulus. European Journal of Wood and Wood Products: Volume 72, Issue 5, 563-570. DUCCI F, GERMANI A, JANIN G, PROIETTI R, SIGNORINI G, 2006: Clone selection for wild cherry (Prunus avium L.) with special reference to some traits used. In: BOZZANO M, RUSANEN M, ROTACH P, KOSKELA J (comp.). EUFORGEN Noble Hardwoods Network, Report of the sixth (9-11 June 2002, Alter do Chão, Portugal) and seventh meetings (22-24 April 2004, Arezzo, Italy). International Plant Genetic Resources Institute, Rome, Italy: 53-60. FIORAVANTI M, SIGNORINI G, DUCCI F, 2009: Prunus avium L.: uno studio sull'ereditabilità del colore del legno. (Prunus avium L.: a study on the heritability of the wood color). Act of the III Selvicolture National Congress, Taormina (ME), 2008 October, Vol. 3: 1498-1503. HANSEN C. P. 2002: Application of the Pilodyn in Forest Tree Improvement. Danida Forest Seed Centre.(Technical Note no. 55).

84 D2.1 – Common protocols and reference standards for selected traits and species

ISHIGURI F, MATSUI R, LIZUKA K, YOKOTA S, YOSHIZAWA N, 2008. Prediction of the mechanical properties of lumber by stress-wave velocity and Pilodyn penetration of 36-year-old Japanese larch trees. Holzforschung 66, 275-280. ISIK F, & LI B. L. 2003: Rapid assessment of wood density of live trees using the Resistograph for selection in tree improvement programs. Canadian Journal of Forest Research, 33(12), 2426-2435. Janin, G., Goncalez, J., Ananías, R., Charrier, B., Fernandes da Silva, G., Dilem, A. (2001). Aesthetics appreciation of wood colour and patterns by colorimetry. part 1. Colorimetry theory for the cielab system. Maderas. Ciencia y tecnología. 3(1-2):03-13 (ISSN 0718-221X). Lepoittevin, C., Harvengt, L., Plomion, C. and Garnier-Gere, P. (2012). Association mapping for growth, straightness and wood chemistry traits in the Pinus pinaster Aquitaine breeding population. Tree Genes Genome 8: 113-126. Lepoittevin, C., Rousseau, J.-P., Guillemin, A., Gauvrit, C., Besson, F., Hubert, F., Da Silva Perez, D., Harvengt, L. and Plomion, C. (2011). Genetic parameters of growth, straightness and wood chemistry traits in Pinus pinaster. Ann For Sci 68: 873-884. MARKUSSEN T, FLADUNG M, ACHERE V, FAVRE JM, ARAGONES A, DA SILVA PEREZ D, HARVENGT L, RITTER E, 2003: Quantitative trait loci controlling growth, chemical and physical wood properties of Pinus pinaster (ait.). Silvae Genetica 52: 8-15. MEIER AGM, SANCHEZ L, SALDA GD, PASTORINO MJM, GAUTRY J-Y, GALLO LA, ROZENBERG P, 2008: Genetic control of the tree-ring response of douglas-fir (Pseudotsuga menziesii (mirb.) franco) to the 2003 drought and heat-wave in France. Ann For Sci 65: 102. Moreau, J. (2010). Impact de pratiques sylvicoles intensives sur les proprietes du bois de pin maritime. (Impact of silviculture on maritime pine wood properties).PhD Thesis, Bordeaux University, pages (in French). (http://ori-oai.u- bordeaux1.fr/pdf/2010/MOREAU_JEROME_2010.pdf). Paiva, J. A. P., Garces, M., Alves, A., Garnier-Gere, P., Rodrigues, J. C., Lalanne, C., Porcon, S., Le Provost, G., Da Silva Perez, D., Brach, J., Frigerio, J.-M., Claverol, S., Barros, A., Fevereiro, P. and Plomion, C. (2008). Molecular and phenotypic profiling from the base to the crown in maritime pine wood-forming tissue. New Phytologist 178: 283-301. Paiva, J. A. P., Garnier-Gere, P. H., Rodrigues, J. C., Alves, A., Santos, S., Graca, J., Le Provost, G., Chaumeil, P., Da Silva-Perez, D., Bosc, A., Fevereiro, P. and Plomion, C. (2008). Plasticity of maritime pine (Pinus pinaster) wood-forming tissues during a growing season. New Phytologist 179: 1080-1094. Pot, D., Chantre, G., Rozenberg, P., Rodrigues, J. C., Jones, G. L., Pereira, H., Hannrup, B., Cahalan, C. and Plomion, C. (2002). Genetic control of pulp and timber properties in maritime pine (Pinus pinaster Ait.). Annals of Forest Science 59: 563-575. Pot, D., Rodrigues, J. C., Rozenberg, P., Chantre, G., Tibbits, J., Cahalan, C., Pichavant, F. and Plomion, C. (2006). Qtls and candidate genes for wood properties in maritime pine (Pinus pinaster Ait.). Tree Gen Genome 2: 10-24. RIGGIO M, ANTHONY RW, AUGELLI F, KASAL B, LECHNER T, MULLER W, TANNERT T, 2014: In situ assessment of structural timber using non-destructive techniques. Materials and Structures/Materiaux et Constructions 47 (5): 749-766. RODRIGUES J, ALVES A, PEREIRA H, SILVA-PEREZ D, CHANTRE G, SCHWANNINGER M, 2006: NIR PLSR results obtained by calibration with noisy, low-precision reference values: Are the results acceptable? Holzforschung 60: 402-408. RODRIGUES J, FUJIMOTO T, SCHWANNINGER M, TSUCHIKAWA S, 2013: Prediction of wood density using NIR- based PLS-R models calibrated with X-ray microdensity. NIR News 24 (2): 4-8.

85 D2.1 – Common protocols and reference standards for selected traits and species

Sandak J, Sandak A, Cantini C, Autino A, 2014: Differences in wood properties of Picea abies L. Karst. in relation to site of provenance and population genetics. Holzforshung (DOI: 10.1515/hf-2014- 0061). SANTONI I, CALLONE E, SANDAK A, SANDAK J, DIRÈ S, 2014: Solid state NMR and IR characterization of wood polymer structure in relation to tree provenance. Carbohydrate Polymers (accepted). SANTOS AJA, ANJOS O, SIMÕES R, RODRIGUES J, PEREIRA H, 2014:Kappa number prediction of Acacia melanoxylon unbleached kraft pulps using NIR-PLSR models with a narrow interval of variation. BioResources 9 (4): 6735-6744. SANTOS A, ALVES A, SIMÕES R, PEREIRA H, RODRIGUES J, SCHWANNINGER M, 2012: Estimation of wood basic density of Acacia melanoxylon (R. Br.) by NIR spectroscopy. Journal of Near Infrared Spectroscopy 20: 267-274. SOUSA-CORREIA C, ALVES A, RODRIGUES J, FERREIRA-DIAS S, ABREU JM, MAXTED N, FORD-LLOYD B, SCHWANNINGER M, 2007: Oil content estimation of single kernel of Quercus sp. acorns by near infrared (NIR) spectroscopy and partial least squares regression (PLSR). Journal of Near Infrared Spectroscopy 15: 247-260. SCHWANNINGER M, RODRIGUES J, FACKLER K, 2011: Band assignments in near-infrared (NIR) spectra of wood and wood components. Journal of Near Infrared Spectroscopy 19: 287-308. SCHWANNINGER M, STEFKE B, HINTERSTOISSER B, 2011: Qualitative assessment of acetylated wood with infrared spectroscopic methods. J Near Infrared Spec. 19 (5): 349-357. SCHWANNINGER M, RODRIGUES J, HINTERSTOISSER B, 2011: Determination of lignin content in Norway spruce wood by Fourier transformed near infrared spectroscopy and partial least squares regression analysis. Part 2: Development and evaluation of the final model. Journal of Near Infrared Spectroscopy 19: 331-341. SCHWANNINGER M, RODRIGUES J, HINTERSTOISSER B, 2011: Determination of lignin content in Norway spruce wood by Fourier transformed near infrared spectroscopy and partial least squares regression analysis. Part 1. Wavenumber-selection and evaluation of the selected range. Section Wood Special Issue 2. Journal of Near Infrared Spectroscopy 19: 319-329. SILVA-PEREZ D, GUILLEMAIN A, ALAZARD P, PLOMION C, ROZENBERG P, RODRIGUES J, ALVES A, CHANTRE G, 2007: Improvement of Pinus pinaster wood elite trees selection by combining near infrared spectroscopy and genetic tools. Holzforschung 61: 611-622. TANNERT T, ANTHONY RW, KASAL B, KLOIBER M, PIAZZA M, RIGGIO M, RINN F, WIDMANN R, YAMAGUCHI N, 2014: In situ assessment of structural timber using semi-destructive techniques. Materials and Structures/Materiaux et Constructions 47 (5): 767-785. WANG X. 2013: Acoustic measurements on trees and logs: A review and analysis. Wood Science and Technology 47(5): 965-975.

86 D2.1 – Common protocols and reference standards for selected traits and species

4.2 Drought resistance

Maria Cristina Monteverdi (CRA) (comp.)

Introduction The report of the IPCC in 2007 confirms that in the last decades the climate is changing, significantly affecting the natural resources on Earth. Several studies have shown how climate change may affect the thermal regimes and precipitation, they are changing very important physiological processes of ecosystems on our planet. According to the IPCC report (2007), although the effect space-time climate change is still partially uncertain, it seems certain that the adaptability / resilience of ecosystems, including forests, is at serious risk. Hence the importance of studying the adaptive biodiversity in relation to climate change at the inter - intraspecific of forest species, in order to identify adaptive traits useful for the sustainable management of genetic resources. Most climate predictive models show increased temperature trends of, and decreased precipitation trends with, a consequent increase of evaporation rate. So the drought stress is one of important abiotic factor that affect natural ecosystems, also forest ecosystems. Therefore, to assess the adaptive traits at inter – intraspecific levels, they play an important role to identify early selection parameters, suitable for new breeding programs of forest reproductive material, able to tolerate the drought condition maintaining a good productivity. All of these tools help to produce forest reproductive material suitable for a sustainable forest ecosystem management. The purpose of this work is to find out a reference method for a joint evaluation that could be identified and shared by scientific community. In particular, the purpose is to obtain information on existing methods to assess drought resistance on their quantitative, qualitative, and operational significance and to identify eventual gaps. A “Survey on different methods to assess drought resistance and tolerance and related adaptive traits” was created and sent to participants:

Some authors defined (LARCHER 1980, TESCHE, 1992) the drought resistance, such as the ability of a plant to ensure its life processes, as keeping its water balance positive during a period of drought as long as possible. In the following, the term of drought resistance will be used as a generic term to describe the ability of tree plants to tolerate, resist, and avoid different levels of water stress.

87 D2.1 – Common protocols and reference standards for selected traits and species

STRESS RESISTANCE (LEVITT 1972)

Stress avoidance Tolerance (real resistance to stress)

Stress avoidance/escape Avoidance of strain to stress Tolerance of strain caused by (ability to avoid stress) For example Drought stress stress Tey are characters -Es: maintaining cell turgor Sample: desert plants that incorporated into the through osmotic reach extremely low values of genotype, but they are not adjustments or cell wall water potential and continue really resistant plants. elasticity the work (the presence of a Examples: little water in the tissues, but - Ability of some plants to lose the ability to maintain active their leaves in the case od metabolism with internal water stresss intrinsic mechanisms. For - effimer plants of the dessert example greater quantity of who make the life cycle only water bound to the plasma when it rains. membrane.)

4.21 Methodology and Evaluation System

The survey is structured into three main parts: A. Overview of parameters, methods, and species investigated. B. Past experiences in drought resistance and tolerance studies (participation to projects, and scientific papers). C. Evaluation of methods according to the following criteria:  objectiveness;  capacity building;  simplicity;  destructiveness;  cost.

4.22 Preliminary results

Participation of involved Institutions Until now, ten Institutions from nine countries answered the questionnaire on a total of 20 participant Institutions on WP2+WP3+Wp11 (Table 4.2.1). Only 10 of 20 participant institutions answered the questionnaire, of these only 4 of 10 are studying or studied drought resistance.

88 D2.1 – Common protocols and reference standards for selected traits and species

Table 4.2.1: Participating institutions involved with the questionnaire, the number of those interested and those that have not answered.

Figure 4.2.2: Results of participation

The geographic distribution of the institutions that answered the questionnaire shows two distinct groups: those involved in the assessment of drought resistance (Mediterranean countries), and those not involved in the study of drought resistance (northern Europe countries).

89 D2.1 – Common protocols and reference standards for selected traits and species

Figure 4.2.3: Geographical distribution of participant institutions that answered to drought resistance questionnaire. The red color indicates the countries involved on drought resistance studies, the blue ones the countries currently not involved on drought resistance studies.

Table 4.2.2: Countries, institutions, and drought resistance interest Countries Respondent institutions Drought resistance interest Austria BOKU - Vienna Yes Netherlands ALTERRA - Wageningen Yes Spain INIA ACoruna Yes Spain CITA - Aragon Yes Italy CRA SEL Yes UK No Germany vTI (now THÜNEN) No France FCBA No Finland METLA (now LUKE) No Belgium VLAGEW No

Overview of parameters, methods, and species investigated From the questionnaire, it appears that the forest species of interest about the drought resistance are the following: Scots pines, black pines, Mediterranean pines, Norway spruce, aspen, beech, ash, Pubescent oak, rowan, maple, oaks, Douglas fir, wild cherry, and silver fir. Table 4.2.3 shows the main objectives of the study declared by involved institutions, with the relative parameters investigated, forest species, and methods used.

90 D2.1 – Common protocols and reference standards for selected traits and species

Objectives Parameters studied Species Method investigated Evaluation of drought Air moisture, soil moisture, Norway spruce Seedlings cultivation in growth chambers effects on biomass physiological drought under different air drought conditions production Individual survival Scots pine, back pine, Watering restriction in nursery /greenhouse Mediterranean pines /phytotron, weighing method, TDR probes

P50 (the applied air pressure causing Norway spruce, Bench top dehydration, 50 % loss of conductivity), aspen, air injection, P80 (the applied air pressure causing beech, acoustic emission testing 80 % loss of conductivity), ash, RWL50 (the applied air pressure pubescent oak, necessary to cause 50 % loss of rowan, relative water content) maple Dry weight ration (leaf, stem, root) Oven and balance (dry at 70 °C for 72 h) Water Use Efficiency (WUE) Mass spectrometer to measure carbon isotopic composition (δ13C)

Photosyntetic parameters (A; gs; T) Gas exchange analysis, portable gas exchange analyser (CIRAS-2, PPSystem) Chlorophyll fluorescence of Portable fluorescence monitoring system photosystem II (PSII) (Hansateck Instruments, FMS 2) Fv/Fm ratio, φPSII (quantum yield of PSII photochemeistry), qP (photochemical quenching), NPQ (non photochemical quenching) Photosynthetic pigments HPLC (High Pressure Liquid Chromatograph) To study the concentration (Chla, Chlb, adaptation to climate xanthophylls cycle) change Antioxidant enzyme activities Enzyme extraction and spectrophotometric assay Oaks, Proline production Proline extraction and determination (BATES Mediterranean pines, et al. 1973 ) spectrophotometer Douglas fir, Phytohormone production Liquid chromatography coupled to tandem wild cherry, mass spectrometry (DURGBANSHI et al. 2005) silver fir Xylem anatomy Microscopic method Root morphology Digital camera, root system analysis software WinRHIZO 4,0 Hydraulic architecture Evaporative flux method (COCHARD et al. 1996) balance, Scholander pressure chamber, sliding microtome

P50 (the applied air pressure causing Cavitron technique (for cavitation 50 % loss of conductivity) vulnerability curves (COCHARD et al. 2005)) Water relations at cell level Free transpiration method and sap expression methods, Scholander pressure chamber Phenological plasticity Scoring system, phenologycal camera (Twingscapes TimelapseCam) Cambial phenology Microscopic method 13 Carbon isotope discrimination ∆ C Extraction methods (BRUGNOLI et al. 1988) (structural carbon and soluble measures carbon isotopic composition sugars), WUE (δ13C), dendro-isotopic analysis Table 4.2.3: Main objectives of the study declared by involved institutions, with the relative parameters investigated, forest species, and methods used

It can be noted overall, that most of the parameters used for the assessment of drought resistance are physiological parameters (64 %), while the quantitative, morphological and phenological parameters are used less, 14 % to 14 % - 9 % respectively.

91 D2.1 – Common protocols and reference standards for selected traits and species

Past experiences in drought resistance and tolerance studies (participation to projects, and scientific papers) The analysis of the results obtained from the questionnaire, indicates that of the institutions that declared they work or have worked on drought resistance, 3 out of 5 have experience in drought resistance and tolerance measurements prior to the year 2000, while 3 out of 5 started studies on drought resistance between 2000 and 2005. Four institutions out of 20 are still studying drought resistance aspects, 1 out of 20 stopped the scientific activities on drought resistance, while 5 out of 20 not actually involved in drought resistance assessments, and more than 11 out of 20 did not answer the questionnaire (Figure 4.2.4). Most of the institutions are involved in international projects and networks, while only a small part is only involved in national programs and projects on drought resistance assessment.

Figure 4.2.4: Distribution of institutions (or groups) in those that are still studying drought resistance aspects, those that have stopped working, those that declared they do not work on drought resistance, and those that have not replied.

Most of the institutions said they don't have specialized staff. About half of institutions have acquired the technical/scientific knowledge on drought resistance by learning by themselves, while a smaller part conducted postgraduate and undergraduate studies, specialized commercial courses in other countries in related subjects.

Methods evaluation Objectiveness of methods The partners involved in the questionnaire indicated 21 different methods for assessing drought resistance. All methods are described in the literature, and almost all are standardized methods according to existing protocols.

92 D2.1 – Common protocols and reference standards for selected traits and species

Capacity building Only 3 out of 21 methods below are easily applicable in the field. Approximately half of below methods require voluminous equipment, and only a few (2 out of 21) need for special infrastructure. Almost all of the below methods (86%) allow statistically valid information to be acquired in a short time. On 21 methods about half allow measurements of multiple parameters to be collected at the same time.

Destructiveness Most of the methods identified are not destructive.

Table 4.2.4: Estimation of destructiveness and/or invasiveness of methods Method Invasive Destructive Bench top dehydration Yes Yes Incremental reaction to reduce air moisture Yes No Watering restriction in nursery/greenhouse/phytotron Yes No Dry weight ratio Yes No Stable isotope analysis Yes No

Photosynthetic pigments analysis (Chla; Chlb; xantophylis cycle) Yes No Antioxidant enzymes activities Yes No Proline and phytohormone production Yes No Xylem anatomy Yes No Root morphology Yes No Hydraulic architecture Yes No Pressure-volume curves Yes No Cavitation curves Yes No Gas exchange method No No Scoring system, phonological camera No No Cambial activity / microscopic method Yes No Extraction of soluble sugars methods (BRUGNOLI et al. 1988), measures No No carbon isotopic composition (δ13C), dendro-isotopic anylysis

Costs The cost analysis is currently in progress due to the absence of direct information.

4.23 Conclusions Currently we can only deduce that the physiological traits are the most investigated to assess the drought resistance. This is probably because of the complexity of information that can be provided, but also for the capacity building of the methods used for their assessment. Normal are methods that allow statistically valid information to be acquired in a short time

93 D2.1 – Common protocols and reference standards for selected traits and species

and are often able to provide simultaneous measurement of multiple parameters. Moreover they are generally not destructive methods. In any case, due to the low number of questionnaires filled out, the results obtained until now do not allow a reference method for a joint evaluation to be identified and shared by scientific community. So these results cannot be considered definitive. In order to get a valid result, the questionnaire should also be sent to the remainder of the project participants and integrated all with the results obtained in TREEBREEDEX project. Otherwise, it could be a good solution to take as reference the results obtained in the TREEBREEDEX project about the reference method for drought resistance assessments.

References

ALIA R, CHAMBEL MR, NOTIVOL E, CLIMENT JM, GONZALEZ-MARTINEZ SC, 2014: Environment-dependent microevolution in a Mediterranean pine (Pinus pinaster Aiton). BMC Genomics, doi: 10.1186/s12862-014-0200-5. CAMARERO JJ, et al., 2003: "The impact of a needleminer (Epinotia subsequana) outbreak on radial growth of silver fir (Abies alba) in the Aragon Pyrenees: A dendrochronological assessment." Dendrochronologia 21 (1): 3-12. JULIO CAMARERO J, et al., 2011: "Synergistic effects of past historical logging and drought on the decline of Pyrenean silver fir forests." Forest Ecology and Management 262 (5): 759-769. CASASOLI M, POT D, PLOMION C, MONTEVERDI MC, BARRENECHE T, LAUTERI M, VILLANI F, 2004: “Identification of QTLs affecting adaptive traits in Castanea sativa Mill.”. Plant Cell and Envirnment (Blasckwell Publications Ltd. Oxford). N. 27: 1088-1101. CENTRITTO M, LAUTERI M, MONTEVERDI MC, SERRAJ R, 2009: Leaf gas Exchange, carbon isotope discrimination and grain yield in contrasting rice genotypes subjected to water deficits during reproductive stage. Journal of Experimental Botany: downloaded from http://jxb.oxfordjournals.org at American College of Thessaloniki on 14 May 2009. pp.1-15. CHAMBEL MR, CLIMENT JM, ALIA R, 2007: Divergence among species and populations of Mediterranean pines in biomass allocation of seedlings grown under two watering regimes. Ann. For. Sci. 64: 87- 97. CLIMENT JM, ALONSO J, GIL L, 2008: Short Note: Root Restriction Hindered Early Allometric Differentiation Between Seedlings of Two Provenances of Canary Island Pine. Silvae Genet. 57: 4– 5. CLIMENT JM, ARANDA I, ALONSO J, PARDOS JA, GIL L, 2006: Developmental constraints limit the response of Canary Island pine seedlings to combined shade and drought. For. Ecol. Manage. 231: 164–168. CLIMENT JM, CHAMBEL MR, LÓPEZ R, MUTKE S, ALIA R, GIL L, 2006: Population divergence for heteroblasty in the Canary Island pine (Pinus canariensis, Pinaceae). Am. J. Bot. 93: 840–8. CLIMENT JM, CHAMBEL MR, PARDOS M, LARIO F, VILLAR-SALVADOR P, 2011: Biomass allocation and foliage heteroblasty in hard pine species respond differentially to reduction in rooting volume. Eur. J. For. Res. 130: 841–850. CLIMENT JM, COSTA E SILVA F, CHAMBEL MR, PARDOS M, ALMEIDA MH, 2009: Freezing injury in primary and secondary needles of Mediterranean pine species of contrasting ecological niches. Ann. For. Sci. 66: 407–415. CLIMENT JM, DANTAS AK, ALIA R, MAJADA J, 2013: Clonal variation for shoot ontogenetic heteroblasty in maritime pine (Pinus pinaster Ait.). Trees 27: 1813–1819.

94 D2.1 – Common protocols and reference standards for selected traits and species

CLIMENT JM, PRADA MA, CALAMA R, CHAMBEL MR, SÁNCHEZ DE RON D, ALIA R, 2008: To grow or to seed: ecotypic variation in reproductive allocation and cone production by young female Aleppo pine (Pinus halepensis, Pinaceae). Am. J. Bot. 95: 833–42. CLIMENT JM, SAN-MARTÍN R, CHAMBEL MR, MUTKE S, 2011: Ontogenetic differentiation between Mediterranean and Eurasian pines (sect. Pinus) at the seedling stage. Trees - Struct. Funct. 175– 186. COCHARD H, BADEL E, HERBETTE S, DELZON S, CHOAT B, JANSEN S, 2013: Methods for measuring plant vulnerability to cavitation: a critical review. J Exp Bot 64: 4779-4791. CORCUERA L, GIL-PELEGRIN E, NOTIVOL E, 2012: Differences in hydraulic architecture between mesic and xeric Pinus pinaster populations at the seedling stage. Tree Physiology 32 (12): 1442-1457. DOI: 10.1093/TREEPHYS/TPS103 CORCUERA L, GIL-PELEGRIN E, NOTIVOL E, 2012: Aridity promotes differences in proline and phytohormone levels in Pinus pinaster populations from contrasting environments. Trees, Structure and Function 26: 799-808. CORCUERA L, COCHARD H, GIL-PELEGRIN E, NOTIVOL E, 2011: Phenotypic plasticity in mesic populations of Pinus pinaster improves vulnerability to xylem embolism (P50) under severe drought. Trees, Structure and Function 25: 1033-1042. CORCUERA L, GIL-PELEGRIN E, NOTIVOL E, 2011: Intraspecific Variation in Pinus Pinaster PSII Photochemical Efficiency in Response to Winter Stress and Freezing Temperatures. PLoS ONE 6(12): e28772. DOI:10.1371/JOURNAL.PONE.0028772 CORCUERA L, GIL-PELEGRIN E, NOTIVOL E, 2010: Phenotypic plasticity in Pinus pinaster δ 13C: environment modulates genetic variation. Annals of Forest Science 67: 812-823. CORCUERA L, CAMARERO JJ, SISÓ S, GIL-PELEGRÍN E, 2006: Radial-growth and wood anatomical changes in overaged Quercus pyrenaica coppice stands: functional responses in a new Mediterranean landscape. Trees: Structure and Function 20: 91-98. CORCUERA L, MORALES F, ABADÍA A, GIL-PELEGRÍN E, 2005: Seasonal changes in photosynthesis and photoprotection in a Quercus ilex subsp. ballota woodland located in its upper altitudinal extreme in the Iberian Peninsula. Tree Physiology 25: 599-608. CORCUERA L, MORALES F, ABADÍA A, GIL-PELEGRÍN E, 2005: The effect of low temperatures on the photosynthetic apparatus of Quercus ilex ssp ballota at its lower and upper altitudinal limits in the Iberian Peninsula and during a single freezing-thawing cycle. Trees: Structure and Function 19: 99- 108. CORCUERA L, CAMARERO JJ, GIL-PELEGRÍN E, 2004: Effects of a severe drought on Quercus ilex radial growth and xylem anatomy. Trees: Structure and Function 18: 83-98. CORCUERA L, CAMARERO JJ, GIL-PELEGRÍN E, 2004: Effects of a severe drought on growth and wood- anatomical properties of Quercus faginea. IAWA Journal 25: 185-204. CORCUERA L, CAMARERO JJ, GIL-PELEGRÍN E, 2002: Functional groups in Quercus species derived from the analysis of pressure-volume curves. Trees: Structure and Function 16: 465-472. DAWALIBI V, MONTEVERDI M.C, MOSCATELLO S, BATTISTELLI A, VALENTINI R, 2014: Effect of salt and water stress on growth, physiological and biochemical responses of two Tamarix spp. accepted the 16- 10-2014 in IForest - Biogeosciences and Forestry EICHERT T, et al., 2010: "Effects of iron chlorosis and iron resupply on leaf xylem architecture, water relations, gas exchange and stomatal performance of field-grown peach (Prunus persica)." Physiologia Plantarum 138 (1): 48-59. ESTESO-MARTINEZ J, et al., 2006: "Competitive effects of herbs on Quercus faginea seedlings inferred from vulnerability curves and spatial-pattern analyses in a Mediterranean stand (Iberian System, northeast Spain)." Ecoscience 13 (3): 378-387.

95 D2.1 – Common protocols and reference standards for selected traits and species

ESTESO-MARTINEZ J, et al., 2006: "Crown architecture and leaf habit are associated with intrinsically different light-harvesting efficiencies in Quercus seedlings from contrasting environments." Annals of Forest Science 63 (5): 511-518. ESTESO-MARTINEZ J, GIL-PELEGRIN E, 2004: "Frost resistance of seeds in Mediterranean oaks and the role of litter in the thermal protection of acorns." Annals of Forest Science 61 (5): 481-486. FARINAS MD, et al., 2012: Shear Waves in Plant Leaves at Ultrasonic Frequencies: Shear Properties of Vegetal Tissues. FARINAS MD, et al., 2013: "Shear waves in vegetal tissues at ultrasonic frequencies." Applied Physics Letters 102 (10). DOLORES FARINAS M, et al., 2014: "MONITORING PLANT RESPONSE TO ENVIRONMENTAL STIMULI BY ULTRASONIC SENSING OF THE LEAVES." Ultrasound in Medicine and Biology 40 (9): 2183-2194. FERNANDEZ V, et al., 2014: "Wettability, Polarity, and Water Absorption of Holm Oak Leaves: Effect of Leaf Side and Age." Plant Physiology 166 (1): 168-180. GARCIA-CEBRIAN F, et al., 2003: "Influence of cotyledon removal on early seedling growth in Quercus robur L." Annals of Forest Science 60(1): 69-73. GRIVET D, CLIMENT JM, ZABAL-AGUIRRE M, NEALE DB, VENDRAMIN GG, GONZÁLEZ-MARTÍNEZ SC, 2013: Adaptive evolution of Mediterranean pines. Mol. Phylogenet. Evol. 68: 555–66. Elsevier Inc. GOMEZ ALVAREZ-ARENAS T, et al., 2009: "Determination of Plant Leaves Water Status using Air-Coupled Ultrasounds." 2009 IEEE International Ultrasonics Symposium: 771-774. GOMEZ ALVAREZ-ARENAS TE, et al. 2009: "Noncontact and noninvasive study of plant leaves using air- coupled ultrasounds." Applied Physics Letters 95 (19). GOMEZ ALVAREZ-ARENAS T E, et al., 2009: "Noncontact and noninvasive study of plant leaves using air- coupled ultrasounds." Applied Physics Letters 95(19): 193702 (193703 pp.)-193702 (193703 pp.). HERNÁNDEZ-SERRANO A, VERDÚ M, SANTOS-DEL-BLANCO L, CLIMENT JM, GONZÁLEZ-MARTÍNEZ SC, PAUSAS JG, 2014: Heritability and quantitative genetic divergence of serotiny, a fire-persistence plant trait. Ann. Bot. 114: 571–7. HIETZ P, ROSNER S, SORZ J, MAYR S, 2008: Comparison of methods to quantify loss of hydraulic conductivity in Norway spruce. Ann. For. Sci. 65, 502p7. HIMRANE H, et al., 2004: "Morphological and ecophysiological variation of the hybrid oak Quercus subpyrenaica (Q. faginea × Q. pubescens)." Trees-Structure and Function 18 (5): 566-575. IPCC, 2007: Climate Change 2007: Synthesis Report. Contribution of Working Groups I, II and III to the Fourth Assessment. Report of the Intergovernmental Panel on Climate Change [Core Writing Team, PACHAURI RK, REISINGER A (eds.)]. IPCC, Geneva, Switzerland, 104 pp. JAVIER PEGUERO-PINA J, et al., 2008: "Frost damage in Pinus sylvestris L. stems assessed by chlorophyll fluorescence in cortical bark chlorenchyma." Annals of Forest Science 65 (8). JAVIER PEGUERO-PINA J, et al., 2009: "Differential photosynthetic performance and photoprotection mechanisms of three Mediterranean evergreen oaks under severe drought stress." Functional Plant Biology 36 (5): 453-462. JAVIER PEGUERO-PINA J, et al., 2011: "Embolism induced by winter drought may be critical for the survival of Pinus sylvestris L. near its southern distribution limit." Annals of Forest Science 68 (3): 565-574. JAVIER PEGUERO-PINA J, et al, 2011: "Hydraulic traits are associated with the distribution range of two closely related Mediterranean firs, Abies alba Mill. and Abies pinsapo Boiss." Tree Physiology 31 (10): 1067-1075. JAVIER PEGUERO-PINA J, et al., 2012: "Leaf anatomical properties in relation to differences in mesophyll conductance to CO2 and photosynthesis in two related Mediterranean Abies species." Plant Cell and Environment 35 (12): 2121-2129.

96 D2.1 – Common protocols and reference standards for selected traits and species

JAVIER PEGUERO-PINA J, et al., 2013: "Three pools of zeaxanthin in Quercus coccifera leaves during light transitions with different roles in rapidly reversible photoprotective energy dissipation and photoprotection." Journal of Experimental Botany 64 (6): 1649-1661. JAVIER PEGUERO-PINA J, et al., 2014: "Morphological and physiological divergences within Quercus ilex support the existence of different ecotypes depending on climatic dryness." Annals of Botany 114 (2): 301-313. LARCHER W, 1980: Ökologie der Pflanzen. 3. Aufl. Ulmer-Verlag, Stuttgart. LAUTERI M, MONTEVERDI MC, SANSOTTA A, CHERUBINI M, SPACCINO L, VILLANI F, KÜÇÜK M, 1999: Adaptation to drought in European chestnut. Evidences from a hybrid zone and from controlled crosses between drought and wet adapted populations. Acta Hort. (ISHS) 494:345-354 http://www.actahort.org/books/494/494_53.htm LAUTERI M, PLIURA A, MONTEVERDI MC, BRUGNOLI E, VILLANI F, ERIKSSON G, 2004: “Genetic variation in carbon isotope discrimination in six European populations of Castanea sativa Mill. Originating from contrasting localities”. Journal of Evolutionary Biology (Blackwell Publishing Ltd). N.17: 1286- 1296. LAUTERI M, MONTEVERDI MC, SCARASCIA MUGNOZZA G, 2010: Preservation of Chestnut (Castanea sativa Mill.) Genetic Resources and Adaptive Potential in Relation to Environmental Changes. Ed. G. Bounous and G.L.Beccaro Acta Hort. 866: 677-682, ISHS 2010 LAUTERI M, HAWORTH M, SERRAJ R, MONTEVERDI MC, CENTRITTO M, 2014: ”Photosynthetic Diffusional Constraints Affect Yield in Drought Stressed Rice Cultivars during Flowering.” PLoS ONE 9(10): e109054. doi:10.1371/journal.pone.0109054. LÓPEZ R, CLIMENT JM, Gil L, 2008: From desert to cloud forest: the non-trivial phenotypic variation of Canary Island pine needles. Trees-Structure Funct. 22: 843–849. LÓPEZ R, CLIMENT JM, Gil L, 2009: Intraspecific variation and plasticity in growth and foliar morphology along a climate gradient in the Canary Island pine. Trees 24: 343–350. LÓPEZ R, ZEHAVI A, CLIMENT JM, GIL L, 2007: Contrasting ecotypic differentiation for growth and survival in Pinus canariensis (Pinaceae). Aust. J. Bot. 55: 1–11. LUIS VC, PUÉRTOLAS J, CLIMENT JM, PETERS J, GONZÁLEZ-RODRÍGUEZ ÁM, MORALES D, et al., 2009: Nursery fertilization enhances survival and physiological status in Canary Island pine (Pinus canariensis) seedlings planted in a semiarid environment. Eur. J. For. Res. 128: 221–229. MAYR S, ROSNER S, 2011: Cavitation in dehydrating xylem of Picea abies: energy properties of ultrasonic emissions reflect tracheid dimensions. Tree Physiology 31: 59-67. MONTEVERDI MC, LAUTERI M, VALENTINI R: “Biodiversity of plant species and adaptation to drought and salt conditions. Selection of species for sustainable reforestation activity to combat desertification.” In Biosaline Agriculture and High Salinity Tolerance. Abdelly, Ozturk, Ashraf, Grignon (eds). Birkhause Verlag AG Basel Switzerland. pp 197-206. PARDOS M, CALAMA R, CLIMENT JM, 2009: Difference in cuticular transpiration and sclerophylly in juvenile and adult pine needles relates to the species-specific rates of development. Trees - Struct. Funct. 23: 501–508. PARDOS M, CLIMENT JM, ALMEIDA H, CALAMA R, 2014: The role of developmental stage in frost tolerance of Pinus pinea L. seedlings and saplings. Ann. For. Sci. 71: 551–562. PEGUERO-PINA JJ, et al., 2007: "Physiological performance of silver-fir (Abies alba Mill.) populations under contrasting climates near the south-western distribution limit of the species." Flora 202 (3): 226-236. PEGUERO-PINA JJ, et al., 2008: "Photochemistry, remotely sensed physiological reflectance index and de-epoxidation state of the xanthophyll cycle in Quercus coccifera under intense drought." Oecologia 156 (1): 1-11.

97 D2.1 – Common protocols and reference standards for selected traits and species

PEGUERO-PINA JJ, et al., 2009: Photosystem II efficiency of the palisade and spongy mesophyll in Quercus coccifera using adaxial/abaxial illumination and excitation light sources with wavelengths varying in penetration into the leaf tissue. Photosynthesis Research 99(1): 49-61. PEMAN J, et al., 2006: "Morphological and functional variability in the root system of Quercus ilex L. subject to confinement: consequences for afforestation." Annals of Forest Science 63 (4): 425- 430. POORTER H, BÜHLER J, DUSSCHOTEN D VAN, CLIMENT JM, POSTMA J, 2012: Pot size matters: a meta-analysis of the effects of rooting volume on plant growth. Funct. Plant Biol. 39: 839–850. PRADA E, ALIA R, CLIMENT JM, DÍAZ R, 2014. Seasonal cold hardiness in maritime pine assessed by different methods. Tree Genet. Genomes 10: 689–701. ROSNER S, KLEIN A, WIMMER R, KARLSSON B, 2006: Extraction of features from ultrasound acoustic emissions: a tool to assess the hydraulic vulnerability of Norway spruce trunkwood? New Phytologist 171: 105-116. ROSNER S, KLEIN A, MÜLLER U, KARLSSON B, 2007: Hydraulic and mechanical properties of young Norway spruce clones related to growth and wood structure. Tree Physiology 27, 1165-1178. ROSNER S, KLEIN A, MÜLLER U, KARLSSON B, 2008: Trade-offs between hydraulic and mechanical stress response of mature Norway spruce trunkwood. Tree Physiology 28, 1179-1188. ROSNER S, KARLSSON B, KONNERTH J, HANSMANN C, 2009: Shrinkage processes in standard-size Norway spruce wood specimens with different vulnerability to cavitation. Tree Physiology 29: 1419-1431. ROSNER S, 2013: Hydraulic and biomechanical optimization in Norway spruce trunkwood: A review. IAWA Journal 34: 365-390. ROSNER S, SVĚTLÍK J, ANDREASSEN K, BØRJA I, DALSGAARD L, EVANS R, KARLSSON B, TOLLEFSRUD MM, SOLBERG S, 2014: Wood density as a screening trait for drought sensitivity in Norway spruce. Can. J. For. Res. 44: 154-161. ROTH-NEBELSICK A, et al., 2013: Stomatal encryption by epicuticular waxes as a plastic trait modifying gas exchange in a Mediterranean evergreen species (Quercus coccifera L.). Plant Cell and Environment 36 (3): 579-589. SANCHEZ-GOMEZ D, et al., 2013: Differences in the leaf functional traits of six beech (Fagus sylvatica L.) populations are reflected in their response to water limitation. Environmental and Experimental Botany 87: 110-119. SANCHO-KNAPIK D, et al., 2010: Air-coupled broadband ultrasonic spectroscopy as a new non-invasive and non-contact method for the determination of leaf water status. Journal of Experimental Botany 61 (5): 1385-1391. SANCHO-KNAPIK D, et al., 2011: Microwave L-band (1730 MHz) accurately estimates the relative water content in poplar leaves. A comparison with a near infrared water index (R-1300/R-1450)." Agricultural and Forest Meteorology 151 (7): 827-832. SANCHO-KNAPIK D, et al., 2012: Air-Coupled Ultrasonic Resonant Spectroscopy for the Study of the Relationship Between Plant Leaves' Elasticity and Their Water Content. Ieee Transactions on Ultrasonics Ferroelectrics and Frequency Control 59 (2): 319-325. SANCHO-KNAPIK D, et al., 2013: The reflectivity in the S-band and the broadband ultrasonic spectroscopy as new tools for the study of water relations in Vitis vinifera L. Physiologia Plantarum 148 (4): 512-521. SANTOS-DEL-BLANCO L, BONSER SP, VALLADARES F, CHAMBEL MR, CLIMENT JM, 2013: Plasticity in reproduction and growth among 52 range-wide populations of a Mediterranean conifer: adaptive responses to environmental stress. J. Evol. Biol. 26: 1912–1924. SANTOS-DEL-BLANCO L, CLIMENT JM, 2014: Costs of female reproduction in a conifer tree: a whole-tree level assessment. J. Ecol. 102: 1310–1317.

98 D2.1 – Common protocols and reference standards for selected traits and species

SANTOS-DEL-BLANCO L, CLIMENT JM, GONZÁLEZ-MARTÍNEZ SC, PANNELL JR, 2012: Genetic differentiation for size at first reproduction through male versus female functions in the widespread Mediterranean tree Pinus pinaster. Ann. Bot. 110: 1449–1460. SANTOS-DEL-BLANCO L, NOTIVOL E, ZAS R, CHAMBEL MR, MAJADA J, CLIMENT JM, 2010: Variation of early reproductive allocation in multi-site genetic trials of Maritime pine and Aleppo pine. For. Syst. 19: 381–392. SCARTAZZA A, PROIETTI S, MOSCATELLO S, AUGUSTI A, MONTEVERDI MC, BRUGNOLI E, BATTISTELLI A, 2001: Effect of water shortage on photosynthesis, growth and storage carbohydrate accumulation in walnut (Juglans regia L.). Acta Hort. (ISHS) 544:227-232. http://www.actahort.org/books/544/544_30.htm SCARTAZZA A, LAUTERI M, MONTEVERDI MC, AUGUSTI A, SPACCINO L, BRUGNOLI E, 1998: Carbon isotope discrimination in leaf and stem carbohydrates in upland rice as affected by drought. In: Photosynthesis: mechanisms and effects. Ed. Garab G., Kluwer Academic Publishers, Dordrecht, the Netherlands, pp. 2569-2572. SCARTAZZA A, MONTEVERDI MC, BARTOLONI L, LAUTERI M, SPACCINO L, DE TULLIO M, BRUGNOLI E, 1996: Effects of drought and ABA on carbon isotope discrimination in structural and non-structural carbohydrates in rice. Plant Physiology and Biochemistry (special issue) pp. 261-262. SZCZYGIEŁ K, 1987: Wpływ wilgotności powietrza na niektóre cechy siewek świerka. Influence of air moisture on some features of spruce seedlings. Sylwan 4: 53-64. TESCHE M, 1992: Klimaresistenz. In: LYR H, FIEDLER H-J, TRANQUILLINI W (Hrsg.): Physiologie und Ökologie der Gehölze. Jena, Stuttgart: Gustav Fischer. 620 S. VALERO GALVAN J, et al., 2011: Studies of variability in Holm oak (Quercus ilex subsp ballota Desf. Samp.) through acorn protein profile analysis." Journal of Proteomics 74 (8): 1244-1255. VALERO-GALVAN J, et al., 2013: Physiological and Proteomic Analyses of Drought Stress Response in Holm Oak Provenances. Journal of Proteome Research 12 (11): 5110-5123. VERDÚ M, CLIMENT JM, 2007: Evolutionary correlations of polycyclic shoot growth in Acer (Sapindaceae). Am. J. Bot. 94: 1316–1320. VILAGROSA A, et al., 2003: Cavitation, stomatal conductance, and leaf dieback in seedlings of two co- occurring Mediterranean shrubs during an intense drought. Journal of Experimental Botany 54 (390): 2015-2024. VILAGROSA A, et al., 2003: Suitability of drought-preconditioning techniques in Mediterranean climate. Restoration Ecology 11 (2): 208-216. VILAGROSA A, et al., 2010: Are symplast tolerance to intense drought conditions and xylem vulnerability to cavitation coordinated? An integrated analysis of photosynthetic, hydraulic and leaf level processes in two Mediterranean drought-resistant species. Environmental and Experimental Botany 69 (3): 233-242. WOLKERSTORFER S, ROSNER S, HIETZ P, 2012: An improved method and data analysis for ultrasound acoustic emissions and xylem vulnerability in conifer wood. Physiologia Plantarum 146: 185-191.

99 D2.1 – Common protocols and reference standards for selected traits and species

5. Reference genotypes

Luc Pâques (INRA) (comp.)

Definition In the course of the TREEBREEDEX project, the need to share common reference genotypes for main forest tree species was noted several times. This might be useful for several research purposes. The concept “genotypes” encompasses different types of genetic units: seed stands, seed orchards, full-sib progenies, half-sib progenies and clones. According to research needs, at least three different types of reference genotypes have been identified, directly linked to genetics & breeding activities or more broadly to ecological studies. Type 1 ‘control genotypes’: in the course of their breeding programme, breeders evaluate their own genotypes and candidate new varieties (for example for certification) relative to some controls towards which they rank their genotypes and estimate genetic gains. Comparison of gains is rarely possible at international level because of this lack of common references. Type 2 ‘signal genotypes’: evaluation of some traits is complex because either it relies on subjective scoring (e.g., stem straightness, branching) or it is done relative to known genotypes within a restricted collection of genotypes available in a country (e.g. phenology, disease resistance). ‘Signal’ genotypes would be useful as references along scoring scales (straight vs crooked/ early flushing vs late flushing/ resistant vs sensitive, etc). They would be unavoidable for international evaluation and later on for comparison of results. Type 3 ‘widespread common genotypes’: for some ecological studies, in particular to monitor climate changes impact (phenotypic plasticity study; phenology modelling; pest & disease epidemics monitoring; etc), the creation of a dense grid of sites with the same genotypes, across contrasted ecological conditions through Europe, would be highly beneficial. To be feasible, the creation of this network should be done in a soft, low-cost way: for example in any new field trial, some few trees (how many?) from various genotypes of some species could be systematically planted, additionally to the main experiment. Further description will be given below. To be successful, conditions are that: a) we are able to identify such genotypes for a given set of species of commercial interest, b) we are able to make them available to the scientific community, c) we agree to use them as much systematically as possible.

Objective The objective of this task is to come up with a roadmap to build up this “reference genotype” provider service and hopefully to implement it as far as we can for a couple of species. Steps include:

100 D2.1 – Common protocols and reference standards for selected traits and species

- identification at national levels of existing genotypes with adequate properties and/or of possibilities to identify such genotypes: collection of this information, agreement on a list of suitable ‘reference genotypes’, - identification of national (infra-)structures which could potentially offer this ‘sharing’ service (seed storage facilities, veg. prop. facilities (nurseries, tissue culture/ somatic embryogenesis lab, etc.)): these structures would have the responsibility to store/preserve the material (seed storage, cryoconservation, rootstock hedges…), to mass-produce it (collect seed lot or buy it, veg. prop. clones) and to disseminate it according to needs. Most probably more than one structure/species is wished. - identification of technical, legal and financial conditions to reliably establish such service.

Description of reference genotypes The nature, the characteristics and the use of these reference genotypes will vary according to their objective. Type1: ‘Control’ Type2: ‘Signal’ Type3: ‘Widespread Nature of genotypes Variable according to Preferably clones Preferably clones the species breeding But some other But some other strategy and its level of material (FS…) might material might be advancement: be necessary when necessary when veg. - synthetic varieties veg. prop. is not/hardly prop. is not/hardly from seed orchard (ex. possible possible (FS for conifers) progenies….?) - clones (ex. for veg- prop. based strategy: ex. poplar, wild cherry) - selected seed stands (i.e., starting programme) Characteristics Genotypes of a given Genotypes of a given Genotypes from species, the best species; contrasting several species; from known material pairs of genotypes for different bioclimates given set of traits across Europe ‘Regionalised’ representing extreme (according to bio- scores on evaluation geographic zones) scale material could be ‘Regionalised’ material needed according to could be needed species (ex. Norway according to species spruce) Use In international and In international and Potentially in all new national genetic trials national genetic trials (genetic) trials to be (e.g. evaluation, of all types established, whatever certification trials) the species tested Conditions - mandatory in - mandatory in Recommended international trials international trials - free but - free but recommended in recommended in

101 D2.1 – Common protocols and reference standards for selected traits and species

national trials national trials Volume of plants Like any other genetic A few (2-5?) ramets / A few (5-10?) trees units tested in the trial clone or sibs / family from each species, for a given species (<10?) for a given whatever the main species species tested

Remarks: Whatever the types of reference genotypes, 1) genotypes should be (as) stable (as possible) in their genetic composition (collection only in good flowering years, mixture of crops from several yrs, …) and their genetic conformity guaranteed. Reference DNA samples could be stored at the Repository Centre (AIT) for control. 2) the most straightforward references to implement this service will be genotypes already mass-produced (from seed orchards, commercially propagated clones), certified and commercially available: these should be probably favoured whenever they fit the above requirements (like for type 1 and perhaps type 3 genotypes above).

102