First Report of Root Rot Caused by Phytophthora lacustris on alder (Alnus lusitanica) in Spain.

Alder decline caused by Phytophthora species is considered one of Europe´s most important diseases of natural ecosystems. In Spain, P. x alni, P. uniformis, P. x multiformis and P. plurivora have been detected in association with root and collar rot in riparian alder populations (Pintos et al. 2010, 2012, 2017; Haque et al. 2014). During the active growth periods 2020-2021, a field survey of the newly designed species Alnus lusitanica (Vít et al. 2017) was carried out at the most symptomatic areas along the Miño-Sil river basin in Galicia and León (NW-Spain). Samples of bark, root, rizosphere soil of declining trees, and river water were collected. Symptoms, similar to those caused by other Phytophthora species, included low leaf density of the canopy, generally with smaller and chlorotic leaves, and branches with dry tips. In some cases, the presence of cankers on the trunk were observed (Figure 1). Necrotic lesions were transferred onto V8-PARPH, a Phytophthora selective medium. Soil and water were baited with carnation petals. Three isolates of one Phytophthora species was recovered: two from the roots of trees, and one from river water at three different sites. The isolates were transferred to V8 agar and incubated a 22ºC in the dark. Colonies had petaloid patterns, and their optimum growth temperature ranged from 25 to 30 °C. In soil extract, non-caducous, non-papillate and ovoid sporangia were produced. Sporangia averaged 41.0×29.4 µm with a length/breadth ratio of 1.39. Internal proliferation occurred (Figure 2). No sexual structures were observed . Genomic DNA was extracted from mycelium obtained from pure cultures of three Phytophthora isolates. The ITS region of the ribosomal DNA template was performed by nested PCR using DC6 (Cooke et al. 2000) and ITS4 (White et al. 1990) in the first round, and ITS6 (Cooke et al. 2000) and ITS4 in the second. The mitochondrial gene cox1 was amplified with primers CoxF4N and CoxR4N (Kroon et al. 2004). BLASTn analyses showed 100% identity to the type of P. lacustris (AF266793, 156 matching bp) for ITS, and 100% identity to the ex-type of P. lacustris (MH136916, 596 matching bp) for the cox1 sequence. Phylogenetic analysis indicated that our isolates were localized in the same evolutionary branch as P. lacustris based on consense sequences of ITS and cox1 sequences. The ITS and cox1 sequences generated in this study were deposited in GenBank with accession number OP588369 and OP548051, and one isolate isolated from root (CECT 21212) was submitted to the Spanish Type Culture Collection (Paterna, Valencia). Pathogenicity tests with isolate CECT 21212 were conducted on ten 3-year-old alders (A. lusitanica) growing in free draining 5 L pots. One shallow cut was made into the cambium at the root collar level. A colonized 5-mm mycelial agar plug, from a 7-day-old culture was inserted into every wound (mycelial surface face-down) and sealed with Parafilm®. Five control plants were inoculated with a sterile agar plug. Plants were kept in a controlled chamber at 25 ºC and 80% humidity. After a 3-week incubation period, inoculated plants showed dieback symptoms and necrosis of the inner bark tissue (Figure 3). Lesion lengths ranged from 2 to 8 cm. Control plants remained symptomless. P. lacustris was recovered from all inoculated plants, but not from the control ones. To our knowledge, this is the first report of P. lacustris causing root rot on alders in Spain. This new detection represents an increased threat to riparian alders by the presence of an additional Phytophthora species associated with alder decline, since P. lacustris can readily adapt to a wide variety of climatic conditions with the ability to infect different hosts (Nechwatal et al. 2013).

Tree growth response to drought partially explains regional-scale growth and mortality patterns in Iberian forests.

Tree-ring data has been widely used to inform about tree growth responses to drought at the individual scale, but less is known about how tree growth sensitivity to drought scales up driving changes in forest dynamics. Here, we related tree-ring growth chronologies and stand-level forest changes in basal area from two independent data sets to test if tree-ring responses to drought match stand forest dynamics (stand basal area growth, ingrowth, and mortality). We assessed if tree growth and changes in forest basal area covary as a function of spatial scale and tree taxa (gymnosperm or angiosperm). To this end, we compared a tree-ring network with stand data from the Spanish National Forest Inventory. We focused on the cumulative impact of drought on tree growth and demography in the period 1981-2005. Drought years were identified by the Standardized Precipitation Evapotranspiration Index, and their impacts on tree growth by quantifying tree-ring width reductions. We hypothesized that forests with greater drought impacts on tree growth will also show reduced stand basal area growth and ingrowth and enhanced mortality. This is expected to occur in forests dominated by gymnosperms on drought-prone regions. Cumulative growth reductions during dry years were higher in forests dominated by gymnosperms and presented a greater magnitude and spatial autocorrelation than for angiosperms. Cumulative drought-induced tree growth reductions and changes in forest basal area were related, but initial stand density and basal area were the main factors driving changes in basal area. In drought-prone gymnosperm forests, we observed that sites with greater growth reductions had lower stand basal area growth and greater mortality. Consequently, stand basal area, forest growth, and ingrowth in regions with large drought impacts was significantly lower than in regions less impacted by drought. Tree growth sensitivity to drought can be used as a predictor of gymnosperm demographic rates in terms of stand basal area growth and ingrowth at regional scales, but further studies may try to disentangle how initial stand density modulates such relationships. Drought-induced growth reductions and their cumulative impacts have strong potential to be used as early-warning indicators of regional forest vulnerability.

Forest resilience to drought varies across biomes.

Forecasted increase drought frequency and severity may drive worldwide declines in forest productivity. Species-level responses to a drier world are likely to be influenced by their functional traits. Here, we analyse forest resilience to drought using an extensive network of tree-ring width data and satellite imagery. We compiled proxies of forest growth and productivity (TRWi, absolutely dated ring-width indices; NDVI, Normalized Difference Vegetation Index) for 11 tree species and 502 forests in Spain corresponding to Mediterranean, temperate, and continental biomes. Four different components of forest resilience to drought were calculated based on TRWi and NDVI data before, during, and after four major droughts (1986, 1994-1995, 1999, and 2005), and pointed out that TRWi data were more sensitive metrics of forest resilience to drought than NDVI data. Resilience was related to both drought severity and forest composition. Evergreen gymnosperms dominating semi-arid Mediterranean forests showed the lowest resistance to drought, but higher recovery than deciduous angiosperms dominating humid temperate forests. Moreover, semi-arid gymnosperm forests presented a negative temporal trend in the resistance to drought, but this pattern was absent in continental and temperate forests. Although gymnosperms in dry Mediterranean forests showed a faster recovery after drought, their recovery potential could be constrained if droughts become more frequent. Conversely, angiosperms and gymnosperms inhabiting temperate and continental sites might have problems to recover after more intense droughts since they resist drought but are less able to recover afterwards.

Do changes in spring phenology affect earlywood vessels? Perspective from the xylogenesis monitoring of two sympatric ring-porous oaks.

This study addresses relationships between leaf phenology, xylogenesis, and functional xylem anatomy in two ring-porous oak species, the temperate Quercus robur and the sub-Mediterranean Q. pyrenaica. Earlywood vessel (EV) formation and leaf phenology were monitored in 2012 and 2013. Ten individuals per species were sampled at each of three sites located in NW Iberian Peninsula. EV areas measured on microcore sections were used to calculate the hydraulic tree diameter (Dh ), in order to model relationships to phenology. Thermal requirements were evaluated using growing degree days (GDD). A species-specific timing of growth resumption was found. The onset of EV formation and budburst were associated to a particular GDD in each species. The onset and duration of EV enlargement affected Dh (and EV size) in Q. robur, but hardly in Q. pyrenaica. The relationship between the timings of EV formation and xylem structure appears to be stronger for the temperate oak, whose larger vessels may result from thermal-induced earlier resumption. In contrast, the sub-Mediterranean oak would maintain a more conservative hydraulic architecture under warming conditions.

Earlywood Anatomy Highlights the Prevalent Role of Winter Conditions on Radial Growth of Oak at Its Distribution Boundary in NW Iberia.

We compared climate-growth relationships (1956-2013) of two natural pedunculate oak (Quercus robur L.) stands with different water-holding capacities growing at the species distribution limit of the Mediterranean Region in NW Iberia. For this, tree-ring chronologies of earlywood vessel size (separating the first row from the other vessels) and latewood width were obtained. Earlywood traits were coupled to conditions during dormancy, whereby an elevated winter temperature appears to induce a high consumption of carbohydrates, resulting in smaller vessels. This effect was reinforced by waterlogging at the wettest site, whose correlation to winter precipitation was strongly negative. Soil water regimes caused differences between vessel rows, since all earlywood vessels were controlled by winter conditions at the wettest site, but only the first row at the driest one; radial increment was related to water availability during the previous rather than the current season. This confirms our initial hypothesis that oak trees near their southern distribution boundary adopt a conservative strategy, prioritizing reserve storage under limiting conditions during the growing period. We believe that wood formation is highly dependent on the balance between the previous accumulation of carbohydrates and their consumption to maintain both respiration during dormancy and early spring growth.

Blue is the fashion in Mediterranean pines: New drought signals from tree-ring density in southern Europe.

Long-term records of tree-ring width (TRW), latewood maximum density (MXD) and blue intensity (BI) measurements on conifers have been largely used to develop high-resolution temperature reconstructions in cool temperate forests. However, the potential of latewood blue intensity (LWBI), less commonly used earlywood blue intensity (EWBI), and delta (difference between EWBI and LWBI, dBI) blue intensity in Mediterranean tree species is still unexplored. Here we developed BI chronologies in moist-elevation limits of the most southwestern European distribution of Pinus nigra subsp. salzmanii Arnold. We tested whether BI variables derived from tree rings of black pine are better proxies than ring-width variables to reconstruct long-term changes in climatic factors and water availability. For this we applied correlations and regression analyses with daily and monthly climate data, a spatial and temporal drought index (Standardized Precipitation-Evapotranspiration Index-SPEI) and Vapour Pressure Deficit (VPD), as well as atmospheric circulation patterns: North Atlantic Oscillation (NAO), Southern Oscillation Index (SOI) and Western Mediterranean Oscillation (WeMO). We found a positive relation between black pine growth (RW) and temperature during the winter preceding the growing season. Among all variables LWBI and dBI were found to be more sensitive than TRW to SPEI at low-elevation site, with EWBI series containing an opposite climatic signal. LWBI and dBI were significantly related to June and September precipitation at high-elevation site. Winter VPD was related with higher EWI and LWI series, whereas dBI and EWBI were related with January SOI and February NAO. We confirm the potential of long-term dBI series to reconstruct climate in drought-prone regions. This novel study in combination with other wood anatomical measurements has wide implications for further use of BI to understand and reconstruct environmental changes in Mediterranean conifer forests.

Non-stationary influence of El Niño-Southern Oscillation and winter temperature on oak latewood growth in NW Iberian Peninsula.

The properties of El Niño-Southern Oscillation (ENSO), such as period, amplitude, and teleconnection strength to extratropical regions, have changed since the mid-1970s. ENSO affects the regional climatic regime in SW Europe, thus tree performance in the Iberian Peninsula could be affected by recent ENSO dynamics. We established four Quercus robur chronologies of earlywood and latewood widths in the NW Iberian Peninsula. The relationship between tree growth and the Southern Oscillation Index (SOI), the atmospheric expression of ENSO, showed that only latewood growth was correlated negatively with the SOI of the previous summer-autumn-winter. This relationship was non-stationary, with significant correlations only during the period 1952-1980; and also non-linear, with enhanced latewood growth only in La Niña years, i.e. years with a negative SOI index for the previous autumn. Non-linear relationship between latewood and SOI indicates an asymmetric influence of ENSO on tree performance, biassed towards negative SOI phases. During La Niña years, climate in the study area was warmer and wetter than during positive years, but only for 1952-1980. Winter temperatures became the most limiting factor for latewood growth since 1980, when mean regional temperatures increased by 1°C in comparison to previous periods. As a result, higher winter respiration rates, and the extension of the growing season, would probably cause an additional consumption of stored carbohydrates. The influence of ENSO and winter temperatures proved to be of great importance for tree growth, even at lower altitudes and under mild Atlantic climate in the NW Iberian Peninsula.

Studying global change through investigation of the plastic responses of xylem anatomy in tree rings.

Variability in xylem anatomy is of interest to plant scientists because of the role water transport plays in plant performance and survival. Insights into plant adjustments to changing environmental conditions have mainly been obtained through structural and functional comparative studies between taxa or within taxa on contrasting sites or along environmental gradients. Yet, a gap exists regarding the study of hydraulic adjustments in response to environmental changes over the lifetimes of plants. In trees, dated tree-ring series are often exploited to reconstruct dynamics in ecological conditions, and recent work in which wood-anatomical variables have been used in dendrochronology has produced promising results. Environmental signals identified in water-conducting cells carry novel information reflecting changes in regional conditions and are mostly related to short, sub-annual intervals. Although the idea of investigating environmental signals through wood anatomical time series goes back to the 1960s, it is only recently that low-cost computerized image-analysis systems have enabled increased scientific output in this field. We believe that the study of tree-ring anatomy is emerging as a promising approach in tree biology and climate change research, particularly if complemented by physiological and ecological studies. This contribution presents the rationale, the potential, and the methodological challenges of this innovative approach.

Selecting earlywood vessels to maximize their environmental signal.

The anatomical features of earlywood vessels often reflect information about past climatic conditions. We examined the relationships between mean monthly temperature and mean vessel lumen area (MVA) in various categories of earlywood vessels. Subsets of earlywood vessels of chestnut (Castanea sativa Mill.) were selected from a previously reported dataset based on several progressive size-related procedures. To include all earlywood vessels, the minimum size considered was 10,000 microm2. Changes in the correlations between MVA and the mean air temperature in March are described and discussed. The results show that not all vessels embody the same information. The MVA of a proportion of the largest earlywood vessels in each annual ring was most closely related to March temperature, whereas MVA of the smallest earlywood vessels was better correlated with June temperature. This difference is probably a result of the vessels being formed at different times: early spring for the largest earlywood vessels and later in spring for the smallest earlywood vessels. Analyses combining large and small vessels yielded lower correlations between MVA and monthly temperature. The number, size and distribution of vessels can vary greatly from ring to ring. In making year-to-year comparisons, the best information is provided by observations on vessels of contemporaneous ontogenesis. Criteria for the selection and analysis of vessels in the assessment of temperature during the season of wood formation are proposed and discussed.

Structure and Function of Intra-Annual Density Fluctuations: Mind the Gaps.

Tree rings are natural archives of climate and environmental information with a yearly resolution. Indeed, wood anatomical, chemical, and other properties of tree rings are a synthesis of several intrinsic and external factors, and their interaction during tree growth. In particular, Intra-Annual Density Fluctuations (IADFs) can be considered as tree-ring anomalies that can be used to better understand tree growth and to reconstruct past climate conditions with intra-annual resolution. However, the ecophysiological processes behind IADF formation, as well as their functional impact, remain unclear. Are IADFs resulting from a prompt adjustment to fluctuations in environmental conditions to avoid stressful conditions and/or to take advantage from favorable conditions? In this paper we discuss: (1) the influence of climatic factors on the formation of IADFs; (2) the occurrence of IADFs in different species and environments; (3) the potential of new approaches to study IADFs and identify their triggering factors. Our final aim is to underscore the advantages offered by network analyses of data and the importance of high-resolution measurements to gain insight into IADFs formation processes and their relations with climatic conditions, including extreme weather events.

Suitability of chestnut earlywood vessel chronologies for ecological studies.

• Wood anatomical features measured in dated tree rings have often proven to be of ecological value. However, little is known about the suitability and power of such measurements studied in a year-to-year basis as is done in dendrochronology. • The present work is based on a comparative analysis of 60 dated time-series of earlywood features of chestnut (Castanea sativa) grown in the climatic context of the Southern part of the Swiss Alps. • It has been shown that the earlywood vessel area is a suitable ecological indicator. This variable, although not very sensitive, contains environmental information that is different from that stored in all other ring-width and earlywood features we considered. The vessel size is mainly related to the temperature during two physiologically crucial periods for vessel growth: the end of the previous vegetation period (during reserve storage) and the onset of cambial activity (during cell division and vessel differentiation). • Our work shows that the mean vessel size of the ring-porous chestnut contains ecophysiological information that can be used for research in dendrochronology.

Earlywood vessels of Castanea sativa record temperature before their formation.

The aim of this study was to identify the climatic signal contained in the earlywood vessel size of the ring-porous chestnut (Castanea sativa) and the physiological processes involved in the underlying mechanisms. In order to assign the encoded signal to a specific physiological process, bud phenology and vessel formation were monitored along an elevation transect and chronologies of the size of the first row of earlywood vessels were retrospectively correlated with 40 yr of early spring temperatures. The first vessels appeared in late April to early May, after encoding both a negative temperature signal in February-March (during tree quiescence) and a positive temperature signal in early April (at the time of resumption of shoot growth). We hypothesize that February and March temperatures affect cambial sensitivity to auxin, preconditioning tree responses later in the season. Furthermore, April temperature is related to tree activation whereby new hormone production fosters vessel expansion.