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Massive propagation of cork oak (Quercus suber) individuals tolerant to Phytophthora cinnamomi (Pc) is probably the most important challenge for cork production. Screening for resistance to Pc of ca. 200 seedlings obtained from a single cork oak tree that has survived the epidemic was performed by soil infestation. Twenty months after Pc inoculation, 33 seedlings survived from Pc infection and the four most vigorous seedlings were selected. The plants were forced to produce new shoots under controlled climatic conditions, and the new shoots were used to establish the plants in vitro by axillary budding. High axillary shoot proliferation rates were achieved by culturing the new shoots on Lloyd and McCown (WPM) medium, followed by subculturing for 2 weeks on 0.22 µM benzyladenine (BA) and for 2 weeks further on 0.04 µM BA. Addition of 20 µM silver thiosulphate (STS) increased the proliferation rates and improved the appearance and development of shoots. Rooting rates of 80–100% were obtained by culturing the shoots for 24 or 48 h on Gresshoff and Doy medium with ⅓ macronutrients plus 122.5 µM indole-3-butyric acid and subsequent transfer to root expression medium containing 20 µM STS. The results of this study optimize the micropropagation of a relevant and recalcitrant tree species in forestry.

Root rot affecting holm oak is a cause of high ecological and economic losses in the Iberian Peninsula, highlighting the relevance of developing disease control methods. The aim of this work was to assess the effect of a biological treatment composed of beneficial organisms (Trichoderma complex, T-complex) on holm oak seedlings infected by Phytophthora cinnamomi in two contrasted holm oak ecotypes, one considered highly susceptible (HU) and another considered tolerant to the pathogen (GR). For this purpose, a complete multifactorial test was carried out in a greenhouse, and seedlings were monitored for survival analysis and morphological and physiological attribute evaluation. Mortality began earlier in the susceptible (HU) than in the tolerant (GR) ecotype, and survival showed different trends due to the inoculation with beneficial microorganisms depending on the ecotype of the plants. The tolerant ecotype showed a high survival rate and better response to the treatment with beneficial microorganisms. GLM showed that the main reason for differences between treatments was ecotype, followed by T-complex and irrigation, and a weak interaction between ecotype and P. cinnamomi was found. The linear relationship between photosynthesis (A) and transpiration (Tr) showed an increase in the A/Tr rates for infected and inoculated plants under drought conditions for the GR ecotype. The tolerant ecotype was benefited more by the beneficial microorganism treatment. The understanding of the genetic diversity of Q. ilex and water stress influence on the efficacy of biological treatments against root rot provides useful information to develop environmentally friendly disease control methods to address the holm oak decline.

Oak decline is a general term used for the progressive dieback and eventual mortality of oak trees due to many compounding stressors, typically a combination of predisposing, inciting, and contributing factors. While pinpointing individual causes of decline in oak trees is a challenge, past studies have identified site and stand characteristics associated with oak decline. In this study, we developed a risk map of oak decline for the Daniel Boone National Forest (DBNF), combining GIS, remote sensing (RS), and public reporting (citizen science, CS). Starting with ground reports of decline (CS), we developed a site-scale model (GIS and RS) for oak decline based on four previously identified predisposing factors: elevation, slope, solar radiation, and topographic wetness. We found that areas identified in the model as having a high oak decline risk also reflected areas of observed oak decline (CS). We then optimized and expanded this risk model to the entire range of the DBNF, based on both site characteristics (as piloted for the case study site) and stand inventory data. The stand inventory data (including species composition and age) further improved the model, resulting in a risk map at the landscape level. This case study can serve as a planning tool and highlights the potential usefulness of integrating GIS, remote sensing, and citizen science.

Oak decline presenting symptoms of tracheomycosis have been observed globally over long periods of time. Since the 1990s, oak decline has been considered to be a multifactorial process where abiotic predisposing factors play a significant role. Nevertheless, biotic factors, such as subcortical insects and fungal pathogens, may influence the decline process, as some insect species transmit spores of tracheomycotic pathogens. We investigated biotic agents (subcortical insects, Ophiostoma spp. and Phytophthora spp.) in six declining oak stands located within mesophytic and thermophytic zones in the Czech Republic, where five dry years occurred from 2015 to 2019. The spectrum of insect pests of oak stands was investigated using two methods: reared from logs from each stand and window traps. The presence of Ophiostoma fungi was assessed from cultivated woodblocks; Phytophthora pathogens were isolated from soil samples. In total, 2931 subcortical insect individuals were captured during the 2021 vegetation period. The most species-rich group of the subcortical insect complex involved in oak decline consisted of the families Cerambycidae: Xylotrechus antilope; Curculionidae: Scolytus intricatus, Xyleborus monographus; and Buprestidae: Agrilus biguttatus, A. sulcicollis. The presence of Ophiostoma was detected in all stands with different intensities between tree parts. Several important oak pests were positively correlated with the occurrence of Ophiostoma. On the contrary, from all soil samples, Phytophthora plurivora was found at only one studied oak stand. Based on the results, a new modified model of biotic and abiotic factors involved in the oak decline is proposed.

Holm oak populations are severely affected by oak decline syndrome, and reliable methods of conserving the plant material are required. A vitrification-based cryopreservation method was used for the first time for the long-term conservation of holm oak embryogenic cultures. Successful cryopreservation was achieved after determining the best developmental stage of the somatic embryos used and the optimal incubation period in plant vitrification solution 2 (PVS2). Embryos were recovered from individual nodular embryogenic structures (NES) derived from four embryogenic lines after preculture on a medium containing 0.3 M sucrose, incubation in PVS2 vitrification solution for 15 min at 25 °C and direct immersion in liquid nitrogen (LN). Embryo recovery rates of 16.7–63.3% were obtained after cryostorage for four years in LN. In addition to the embryo developmental stage and the PVS2 treatment time, the genotype can also significantly affect embryo recovery after LN storage. There were no significant differences in plant regeneration or polyploid stability between somatic embryos and plants derived from control embryos (not cryopreserved) and cryopreserved embryos. The findings indicate that embryo proliferation, plant conversion and polyploid stability are maintained in material recovered from the vitrification solution and subsequently cryopreserved.

The dehesa is a traditional agrosilvopastoral ecosystem characterized by a savanna-like structure. It has been in decline since the middle of the last century, in particular in agricultural environments where it has been relegated to isolated forests. This paper presents a study into the reduction over the past 50 years. One of the main causes of this decline has been identified as xylophagous insects, specifically the wood borer Cerambyx welensii Kuster (Coleoptera: Cerambycidae). Based on estimations in both the population size, using Jolly Seber models, and the level of tree injury, using a single-index model, the study investigates the role the insect has played in the decline of the dehesa. Surface area loss was found to be greater than 50%, while canopy cover has diminished linearly over the period studied, with the risk of disappearance of the woodland increasing in the same magnitude as borer population density. The highest wood borer population densities observed corresponded to stands with an average canopy cover loss of 1.68% a year, indicating a risk of completely losing the forest cover in the short term (10–30 years). The proposed tree injury index was successful in categorizing the size of the insect population, and seems to be a useful indicator for describing the relationship between the two variables. According to this indicator, woodlands with more than 10% of trees with severe or very severe injuries are at risk. Follow-up work should be undertaken to investigate long-term demographic changes in wood borer populations, and management practices should be developed to reduce the population size, minimize forest loss and preserve these relict oak trees in unconnected landscapes.

Holm oak populations have deteriorated drastically due to oak decline syndrome. The first objective of the present study was to investigate the use of axillary budding and somatic embryogenesis (SE) to propagate asymptomatic holm oak genotypes identified in disease hotspots in Spain. Axillary budding was achieved in two out of six tolerant genotypes from the south-western region and in two out of four genotypes from the Mediterranean region. Rooting of shoots cultured on medium supplemented with 3 mg L−1 of indole-3-acetic acid plus 0.1 mg L−1 α-naphthalene acetic acid was achieved, with rates ranging from 8 to 36%. Shoot cultures remained viable after cold storage for 9–12 months; this procedure is therefore suitable for medium-term conservation of holm oak germplasm. SE was induced in two out of the three genotypes tested, by using nodes and shoot tips cultured in medium without plant growth regulators. In vitro cloned progenies of the tolerant genotypes PL-T2 and VA5 inhibited growth of Phytophthora cinnamomi mycelia when exposed to the oomycete in vitro. Significant differences in total phenol contents and in the expression profiles of genes regulating phenylpropanoid biosynthesis were observed between in vitro cultured shoots derived from tolerant trees and cultures established from control genotypes.

Oaks are dominant and key tree species in Mediterranean forest ecosystems. However, in recent decades, oak forests have been heavily impacted by oak decline, a worldwide phenomenon exacerbated by climate change. The charcoal disease agent Biscogniauxia mediterranea is involved in the decline of Mediterranean oak formations in a variety of contexts. Here, we investigated the impact and role of B. mediterranea in the decline of oaks in Castelfidardo Forest, a relict wood of the late Holocene and a Site of Community Importance. We established five plots within which we recorded tree positions, any symptoms and signs of decline, association of B. mediterranea to declining trees, and deadwood and associated mycota. Of 471 oaks inspected, 7.0% showed brownish exudates on the stems, 46.9% showed epicormic shoots along the main trunk, and 24.4% showed black carbonaceous stromata on diseased branches and trunks. The decline was most severe for Quercus cerris, which comprised plots #4 and #5, at 50.0% (81/162 trees) and 29.0% (33/114), respectively; then for Quercus robur for plot #3, at 40.0% (38/95); and finally for Quercus pubescens for plots #1 and #2, at 13.7% (7/51) and 12.3% (6/49), respectively. Bark tissues were collected from trees with charcoal cankers and taken to the laboratory for microscopic examination and identification by mycological and molecular methods. This investigation revealed a close association between oaks with pronounced reduction of vitality and incidence of B. mediterranea. Deadwood was equally distributed among the five plots, and was heavily colonized by Basidiomycota. The high incidence of the charcoal canker pathogen B. mediterranea appeared to be related to environmental stresses. However, the absence of silvicultural management, high competition among physiologically mature trees, and the geographic isolation of this residual forest may have predisposed oaks to decline.

We studied the maternally inherited chloroplast DNA polymorphism at three microsatellite loci of 157 Quercus robur trees from 38 native populations in Lithuania. We found high diversity of eight haplotypes from the Balkan lineage A (frequency 0.75) and the “German” subbranch of the Balkan lineage A (freq. 0.12), western and eastern Italian lineages C (freq. 0.05 and 0.06, respectively), and Iberian lineage B (freq. 0.03). The haplotypes were geographically well structured (among population differentiation index PhiPT = 0.30, the p-value < 0.001) that is unexpected for such a small territory as Lithuania. We raised a hypothesis on historical overexploitation of oaks by eliminating certain haplotypes in Lithuania, following a drastic felling of oak forests over the last few centuries.

We present a reproducible procedure for transforming somatic embryos of cork oak with the CsTL1 gene that codes for a thaumatin-like protein, in order to confer tolerance to Phytophthora cinnamomi. Different concentrations/combinations of the antibiotics carbenicillin and cefotaxime, as bacteriostatic agents, and kanamycin, as a selective agent, were tested. A lethal dose of 125 mg/L kanamycin was employed to select transgenic somatic embryos, and carbenicillin was used as a bacteriostatic agent at a concentration of 300 mg/L, which does not inhibit somatic embryo proliferation. The transformation efficiency was clearly genotype-dependent and was higher for the TGR3 genotype (17%) than for ALM80 (4.5%) and ALM6 (2%). Insertion of the transgenes in genomic DNA was confirmed by PCR analysis, whereas expression of the CsTL1 gene was evaluated by semi-quantitative real-time PCR (qPCR) analysis. A vitrification treatment successfully cryopreserved the transgenic lines generated. The antifungal activity of the thaumatin-like protein expressed by the gene CsTL1 was evaluated in an in vitro bioassay with the oomycete P. cinnamomi. Of the eight transgenic lines analyzed, seven survived for between one or two times longer than non-transgenic plantlets. Expression of the CsTL1 gene and plantlet survival days were correlated, and survival was generally greater in plantlets that strongly expressed the CsTL1 gene.

Cork oak forests are of immense importance in terms of economic, cultural, and ecological value in the Mediterranean regions. Since the beginning of the 20th century, these forests ecosystems have been threatened by several factors, including human intervention, climate change, wildfires, pathogens, and pests. Several studies have demonstrated the primary role of the oomycete Phytophthora cinnamomi Ronds in the widespread decline of cork oaks in Portugal, Spain, southern France, and Italy, although other congeneric species have also been occasionally associated. Between 2015 and 2019, independent surveys were undertaken to determine the diversity of Phytophthora species in declining cork oak stands in Sardinia (Italy). Rhizosphere soil samples were collected from 39 declining cork oak stands and baited in the laboratory with oak leaflets. In addition, the occurrence of Phytophthora was assayed using an in-situ baiting technique in rivers and streams located throughout ten of the surveyed oak stands. Isolates were identified by means of both morphological characters and sequence analysis of internal transcribed spacer (ITS) regions of ribosomal DNA. In total, 14 different Phytophthora species were detected. Phytophthora cinnamomi was the most frequently isolated species from rhizosphere soil, followed by P. quercina, P. pseudocryptogea, and P. tyrrhenica. In contrast, P. gonapodyides turned out to be the most dominant species in stream water, followed by P. bilorbang, P. pseudocryptogea, P. lacustris, and P. plurivora. Pathogenicity of the most common Phytophthora species detected was tested using both soil infestation and log inoculation methods. This study showed the high diversity of Phytophthora species inhabiting soil and watercourses, including several previously unrecorded species potentially involved in the decline of cork oak forests.

At the forest landscape/region level, based on annual Forest Inventory and Analysis plot data from 1999 to 2010, oak decline and mortality trends for major oak species (groups) were examined in the Ozark Highlands of Arkansas and Missouri. Oak decline has elevated cumulative mortality of red oak species to between 11 and 15 percent in terms of relative density and basal area of standing dead oak trees, respectively. These values are three to five times higher than for white oak group and non-oak species. Oak decline and associated escalating mortality have occurred primarily in red oak species while the white oak group has maintained a relatively stable mortality rate that is comparable to non-oak species. Cross-correlation analyses indicate that mortality in the red oak group was significantly correlated with the growing season Palmer drought severity index (PDSI) and usually lagged two to three years following single drought events. Moreover, based on the past 17 years PDSI data, it appears that the cumulative impacts of drought may last up to 10 years. The Ozark Highlands experienced a severe drought extending from 1998 to 2000 and another milder drought from 2005 to 2006. These drought events triggered the escalation of mortality starting around year 2000. Spatially, high red oak mortality sites (hot spots with proportional basal area mortality > 0.12) initially occurred in the central Ozarks and spread gradually over most of the Ozark Highlands as regional droughts continued. In contrast, sites with elevated white oak and non-oak mortality occurred sporadically, mainly in the southern portion (Arkansas) of the Ozark Highlands. During the most recent inventory period (2006–2010), over 60%, 7% and 5% of red oak, white oak and non-oak groups, respectively, had relative mortality rates of > 12%.

The sustainability of “dehesas” is threatened by the Holm oak decline. It is thought that the effects of root rot on plant physiology vary depending on external stress factors. Plant growth and biomass allocation are useful tools to characterize differences in the response to drought and infection. The study of physiological responses together with growth patterns will clarify how and to what extent root rot is able to damage the plant. A fully factorial experiment, including drought and Phytophtora cinnamomi Rands infection as factors, was carried out with Quercus ilex L. seedlings. Photosynthesis, biomass allocation and root traits were assessed. Photosynthetic variables responded differently to drought and infection over time. The root mass fraction showed a significant reduction due to infection. P. cinnamomi root rot altered the growth patterns. Plants could not recover from the physiological effects of infection only when the root rot coincided with water stress. Without additional stressors, the strategy of our seedlings in the face of root rot was to reduce the biomass increment and reallocate resources. Underlying mechanisms involved in plant-pathogen interactions should be considered in the study of holm oak decline, beyond the consideration of water stress as the primary cause of tree mortality.

Holm oak populations are severely affected by oak decline syndrome, and reliable methods of conserving the plant material are required. A vitrification-based cryopreservation method was used for the first time for the long-term conservation of holm oak embryogenic cultures. Successful cryopreservation was achieved after determining the best developmental stage of the somatic embryos used and the optimal incubation period in plant vitrification solution 2 (PVS2). Embryos were recovered from individual nodular embryogenic structures (NES) derived from four embryogenic lines after preculture on a medium containing 0.3 M sucrose, incubation in PVS2 vitrification solution for 15 min at 25 °C and direct immersion in liquid nitrogen (LN). Embryo recovery rates of 16.7-63.3% were obtained after cryostorage for four years in LN. In addition to the embryo developmental stage and the PVS2 treatment time, the genotype can also significantly affect embryo recovery after LN storage. There were no significant differences in plant regeneration or polyploid stability between somatic embryos and plants derived from control embryos (not cryopreserved) and cryopreserved embryos. The findings indicate that embryo proliferation, plant conversion and polyploid stability are maintained in material recovered from the vitrification solution and subsequently cryopreserved.

Holm oak is a key tree species in Mediterranean ecosystems, whose populations have been increasingly threatened by oak decline syndrome, a disease caused by the combined action of Phytophthora cinnamomi and abiotic stresses. The aim of the present study was to produce holm oak plants that overexpress the Ginkbilobin-2 homologous domain gene ( Cast&#95;Gnk2-like ) that it is known to possess antifungal properties. Proembryogenic masses (PEMs) isolated from four embryogenic lines (Q8, E2, Q10-16 and E00) were used as target explants. PEMs were co-cultured for 5 days with Agrobacterium EHA105pGnk2 and then cultured on selective medium containing kanamycin (kan) and carbenicillin. After 14 weeks on selective medium, the transformation events were observed in somatic embryos of lines Q8 and E2 and a total of 4 transgenic lines were achieved. The presence of the Cast&#95;Gnk2-like gene on transgenic embryos was verified by PCR, and the number of transgene copies and gene expression was estimated by qPCR. Transgenic plants were obtained from all transgenic lines after cold storage of the somatic embryos for 2 months and subsequent transfer to germination medium. In an in vitro tolerance assay with the pathogen P. cinnamomi , we observed that transgenic plants were able to survive longer than wild type.