1

COST Action FP1106 STReESS: STSM-report

Reference: Short-Term Scientific Mission (STSM), Cost Action FP1106

Reference code: COST-ONLINE_STSM-FP1106-20372

Beneficiary: Angela Balzano, University of Naples Federico II, Dept. Agricultural and Food Sciences, Portici(IT) , angela.balzano@unina.it

STSM Title: "Wood formation in mediterranean hardwood and softwood species: Arbutus unedo and Pinus halepensis"

Hosts: Katarina Cufar,University of Ljubljana, Biotechnical Faculty, Department of Wood Science and Technology,Ljubljana(SI), katarina.cufar@bf.uni-lj.si

Places: Department of Wood Science and Technology, Biotechnical Faculty, University of LjubljanaLjubljana(SI), katarina.cufar@bf.uni-lj.si

Periods: 15 September 2014– 15 December 2014

1. Introduction & Purpose of the STSM

Mediterranean climate is characterized by hot and dry summers alternating with cool and wet winters; the typical vegetation is called “Macchia”. Mediterranean-type of ecosystems, is expected to be susceptible to current climate change with longer periods of drought; more concentrated rainfalls, and increased temperatures. This will promote unprecedented drought stress in vegetation. Plants can be damaged directly and indirectly by drought. Direct damage is made to the cambium activity. To cope with drought conditions, Mediterranean species have evolved morphological adaptations (De Micco et al., 2008), which often result in specific patterns of cambial activity leading to intra-annual density fluctuations (IADFs) in the wood. The occurrence of IADFs makes difficult to date tree rings but such IADFs can provide information about the relationship between environmental factors and eco-physiological processes during tree growth with intra-annual resolution. To understand better IADFs formation and factors affecting it, it is necessary to study seasonal cambial activity.

The proposed STSM project was aimed to acquire methodological expertise to analyse cambial activity in model species and to transfer it to Mediterranean species forming IADFs, namely Arbutus unedo (hardwood) and Pinus halepensis (softwood).

The operational objective was to identify and overcome critical points in the analysis of cambial activity in Mediterranean hardwoods: wood hardness (easily causing breaking of mocrocoring tools) and anatomical complexity.

2

2. Description of the work carried out during the STSM

The main work carried out in Ljubljana was done in the laboratory under the supervision of Prof. Dr. Katarina Čufar and her collaborators Dr. Maks Merela, and Luka Krze as well as Dr. Jožica Griča.

In the first phase (lasting two weeks), I worked on Picea abies and Fagus sylvatica (species routinely analysed at the host institutione) from the sampling in the fild, acquiring skills on all the steps for the methodology to study cambial activity and specifically: sampling, preparation of samples for microscopy (e.g. embedding, cutting) and microscopy analysis of sections. In the second phase (lasting until the end of the STSM), I worked on the microcores of the two Mediterranean species which I previously collected in Italy within the activities of my Doctoral Course. Sampling site (Fig. 1) was selected on the Thyrrhenian Cost, in Quisisana, Castellammare di Stabia (Naples), Southern Italy. Sampling site is located approximately at 400 m a.s.l. and is occupied by typical Macchia characterized by Mediterranean shrub and tree species.

Figure 1. Sampling site, Quisisana, Castellammare di Stabia (Naples), Southern Italy.

Microcores were collected with Trephor (Rossi et al., 2006) from eight trees of Arbutus

unedo and eight trees of Pinus halepensis at breast height, following a spiral. Microcores were fixed immediately in 70% ethanol and stored in eppendorfs at 4 °C until the procedure of sample preparation started. In Ljubljana I prepared the samples following the procedure described in the protocol "Wood and Sample Preparation for Microscopic Analysis" (Prislanet al. 2014). I performed the following steps:

• Identification and marking the transverse plane of microcores.

• Dehydration and infiltration with paraffin in tissue processor LEICA TP 1020© (2002, Leica Microsystem) (Fig. 2a).

• Preparation of paraffin blocks with a paraffin dispenser LEICA EG 1120© (fig. 2b sx) and heatable eletric forceps LEICA EG F© (Fig. 2b dx)

• Reduction of paraffin blocks

• Trimming of the paraffin blocks and cutting of samples with a semi-automatic rotary microtome LEICA RM 2245© (fig. 1c) obtaining traverse cross-sections about 9 µm thick.

• Staining with a water solution of safranin and astra blue (Werf van der et al., 2007)

• Preparation of permanent slides mounted in Euparal (Bioquip)

3

Figure 2. a LEICA TP 1020©, b LEICA EG 1120© and LEICA EG F©, c LEICA RM 2245©

The main constraint I found in sample preparation was linked to the achievement of microsections of high quality. The microcores, in particular those of Arbutus unedo, in some cases were either damaged or with wood and bark apart. Such problems were probably due to the hardness of the wood that had blunt Trephor's blade during the sampling, and effected the quality of the samples. Furthermore, some cracks occurred within cambial zone: this is probably due to the fact that the microcores were stored directly into 70% ethanol obtaining a too abrupt dehydration of the tissues. The quality of the slides was not always perfect, but still good enough to perform image analysis to study cambial activity and xylogenesis. The measurements were performed with a Nikon microscope and 800 Nis Elements BR3 image analysis system software. Many digital microphotographs were captured with Nikon DS-Fi1 digital camera.

I measured wood formation in Pinus halepensis (fig. 3) quantifying:

- Number of Cambial Cells (CC)

- Number of Post Cambial Cells (PC)

- Number of cells with developing Secondary Wall (SW)

- Number of Mature Cells (MT)

- Number of cells in Previous Xylem ring (PX)

- Width of the xylem ring (in micrometers)

- Width of previous 3 xylem rings

Figure 3. Cross-section of P. halepenis with bark, cambial zone and wood. Image was taken with an objective of 10x magnification.

a b c

4

I mesured wood formation in Arbutus unedo (Fig. 4) quantifying:

- Number of Cambial Cells (CC)

- Width (µm) of Cambial zone

- Width of tissue with Post Cambial Cells (PC)

- Width of tissue with developing Secondary Wall (SW)

- Width of tissue with Mature Cells (MT)

-Width of previous 3 xylem rings

Figure 4. Cross-section of A. unedo with bark, cambial zone and wood. Image was taken with an objective of 10x magnification. Once I finished measurements I created graphs representing wood formation of each tree and presented pictures of micro-sections for each date.

3. Description of the main result obtained

I prepared about 200 slices during my STSM, about 100 of Pinus halepensis and about 100 of Arbutus unedo, one set of slices per sampling. I tried to get high-quality slides, following the various steps of the protocol. Good results were obtained with samples of Pinus halepensisby applying the standard protocol. For samples of Arbutus unedo, I met more difficulties related to the hardness of wood. Adopting some adjustments, such as leaving the samples for longer time in water before cutting, or useing a new blade for each sample, I got good sections also in such case. However, some problems were related to sampling carried out with Trephor. Lack of experiences and blunting of the tool caused the problems. During the process of removing micro-core from the tube of the Trephor, compression of the tissues can also cause cell deformation in the meristems, collapse of enlarging cells or cracks in the cell walls (Rossi et al. 2006). To obtain adequate quality, micro-cores should be collected 10 cm apart from each other, following a spiral up the stem to avoid wound effects (Prislan et al. 2011). Gentle pressure (with a needle or toothpick) should be applied to separate the microcore from the cutting tube. Despite the

described problems, slide quality obtained was in most cases good enough to perform image analysis and measurements of wood formation of the two species in the period between June to September as shown in Fig. 5 and Fig. 6. I also had the opportunity to practise with these adjustments in microcore collection.

5

Figure 5. Numbers of CC, PC, SW and MT of Pinus halepensis from June to September are shown in the graph above. Images shown below represent microsections of tissues used to perform image analyses.

6

Figure 6. Numbers of CC, PC, SW and MT of Pinus halepensis from June to September are shown in the graph above. Images shown below represent microsections of tissues used to perform image analyses.

Notwithstanding the shortness of the period and the difficulty to distinguish IADFs from tree-rings, it is possible to hypothesize how the cambium works in this species.

I intend to take the last microcore from every tree in December 2014. I also intend to take cores to carry out dendrochronological analyses. Moreover, I will apply epi-fluorescence microscopy on the obtained microsections to highlight various layers of the cambium and cell differentiation zones. All results could be used for a technical or research publication.

7

4. Description about how the results contribute to the Action aims

This STMS allowed starting the collaboration and supporting the exchange of knowledge between the Department of Agricultural and Food Sciences of the University of Naples where I am Doctoral Student and the Department in Ljubljana where I was hosted. The results of this STSM will contribute to the principal aims of the Action’s FP1106, and main aims of the Working Group I “to establish a platform to survey and integrate information about stress response of treesto extreme events on tree growth, wood anatomy, and ecophysiology and laboratory experiments”, and the Working Group III “to help identifying the existing knowledge gaps on tree response to environmental stress”. The topic of my work fits well with the activities of the Topic groups 2,4 and 5.

5. Confirmation by the host institutions of the successful execution of the STSM

The confirmation letter of the host institution of successful execution of the STSM is attached in the file.

6. Authorization to post the report at the Action website

I agree that this report is posted at the Action website.

7. References

Rossi, S., Anfodillo, T., & Menardi, R. (2006). Trephor: a new tool for sampling microcores from tree stems. Iawa Journal, 27(1), 89.

De Micco, V., Aronne, G., & Baas, P. (2008). Wood anatomy and hydraulic architecture of stems and twigs of some Mediterranean trees and shrubs along a mesic-xeric gradient. Trees, 22(5), 643-655.

Werf van der GW, Sass-Klaassen U, Mohren GMJ (2007) The impact of the 2003 summer drought on the intra-annual growth pattern of beech (Fagus sylvatica L.) and oak (Quercus robur L.) on a dry site in the Netherlands. Dendrochronologia 25:103–112.

Prislan, P., Koch, G., Čufar, K., Gričar, J., & Schmitt, U. (2009). Topochemical investigations of cell walls in developing xylem of beech (Fagus sylvatica L.).Holzforschung, 63(4), 482-490.

Prislan, P., Gričar, J., Čufar, K. (2014). Wood Sample Preparation for Microscopic Analysis.

8