Strain‐related pathogenicity in Diplodia corticola

Diplodia corticola is one of the most aggressive fungal pathogens of Quercus species and is involved in the decline of Mediterranean cork oak forests and Californian oaks. Information regarding variation in virulence between strains is scarce. We hypothesize that D. corticola strains differ in virulence and consequently induce different symptoms in infected plants. To test this, infection assays were carried out on Quercus suber half‐sib seedlings with seven strains of D. corticola. Visual symptoms of infection (external lesions, leaf wilting, exudation and others) were recorded in parallel with physiological and biochemical parameters. All strains were able to cause lesions but at differing levels of aggressiveness. We show that internal lesion length did not correlate directly with strain aggressiveness and this agrees with physiological parameters that should be taken into account to infer about strain pathogenicity. Infection by all strains induced an overall negative impact on the net photosynthetic rate and an increase in the oxidative stress status of plants; however, significant differences were found when the effects of different strains were compared. Results also suggest that being under optimum growth conditions, prior to and during infection, allowed plants to respond to the pathogen. At the end of the experiment, some strains of D. corticola established a latent pathogen‐like relationship with cork oak. This is the first study to show that D. corticola virulence is strain‐dependent.     

Viability of holm and cork oak seedlings from acorns sown in soils naturally infected with Phytophthora cinnamomi

The emergence and survival of pregerminated holm oak (Quercus ilex) and cork oak (Quercus suber) acorns from two ecologically different dehesas (Mediterranean open woodlands) were studied in two soils from these stands naturally infected with Phytophthora cinnamomi, and in the same soils previously sterilized in the autoclave. Phytophthora cinnamomi was consistently isolated from the radicles of all unemerged and all emerged but dead seedlings from the unsterilized substrates. Seedlings of holm oak were more susceptible to P. cinnamomi than those of cork oak. Mortality of holm oak seedlings was significantly different depending only on soil treatment (sterilized or unsterilized), and it was 100% in unsterilized soils, independent of acorn provenance and soil origin. Mortality of cork oak seedlings was significantly different depending on the acorn origin and soil treatment, and on the interactions acorn origin × soil origin and soil origin × soil treatment. The demonstrated high susceptibility of holm and cork oak young seedlings to P. cinnamomi could be a limiting factor in Mediterranean open woodlands (dehesas) not only in natural regeneration processes but also when reforestation by direct sowing is implemented.     

Diplodia corticola and Phytophthora cinnamomi: the main pathogens involved in holm oak decline on Caprera Island (Italy)

Since 2008, severe and widespread tree decline and mortality has been observed at the main growing Quercus ilex L. (holm oak) forest on Caprera Island, Italy. To clarify the symptomatology and aetiology of this phenomenon, field surveys and isolations from symptomatic trees were carried out in summer 2010. Affected trees exhibited crown thinning, branch dieback, sunken cankers, epicormic shoots, exudates on branches and trunk, root losses and sudden death symptoms. Four fungal species belonging to Botryosphaeriaceae family, namely Botryosphaeria dothidea, Diplodia corticola, D. seriata and Neofusicoccum parvum, were isolated from cankers on trunk and branches, whereas three species of Phytophthora, namely P. cinnamomi, P. cryptogea and P. gonapodyides, were isolated from fine roots and rhizosphere soil samples. Isolates were identified using both morphological analysis and DNA‐based techniques. Pathogenicity trials on holm oak seedlings showed that all the isolated species are pathogenic. D. corticola proved to be the most aggressive species. Our results provide the first evidence for a combined involvement of D. corticola and P. cinnamomi in the aetiology of holm oak decline in Italy and suggest that these pathogens are not only important contributing factors in the onset of long‐term tree decline, but also may cause the rapid devastation of extensive oak ecosystems.     

Growth and physiological responses of Quercus brantii seedlings inoculated with Biscogniauxia mediterranea and Obolarina persica under drought stress

Charcoal canker of oak is a common disease in the western regions of Iran, which has widely spread due to climate change and increasing drought in recent years. Biscogniauxia mediterranea and Obolarina persica are known as two agents of oak charcoal canker in Zagros forests causing the dieback of oak trees. The effects of charcoal disease agent pathogens on the growth and physiological response of two‐year‐old seedlings of Quercus brantii were evaluated under drought stress in greenhouse over a period of nine months. Survival was 21.7% lower in seedlings inoculated with B. mediterranea subjected to drought stress compared with control treatment. The length of lesions on stems was 68% more under charcoal pathogens in drought stressed compared with the length of lesions caused by charcoal pathogens only. On the other hand, stem lesion length caused by B. mediterranea was 25% greater than those caused by O. persica. Some morphophysiological characteristics were affected by charcoal pathogens alone and/or in combination with drought. Diameter, height and biomass decreased the most in seedlings inoculated with charcoal pathogens under drought stress. Pathogenic agents alone and in combination with drought stress reduced photosynthetic rates, stomatal conductance, transpiration, the maximum Rubisco activity (V_cmax), maximum photochemical efficiency of photosystem II (F_v/F_m) and xylem water potential. In general, drought increased the aggressiveness of fungus and intensification of their destructive effects in Quercus brantii. Moreover, there was no significant difference between the effect of B. mediterranea and O. persica on the morphophysiological parameters studied.     

Water-use efficiency in cork oak (Quercus suber) is modified by the interaction of water and light availabilities

We studied the interaction of light and water on water-use efficiency in cork oak (Quercus suber L.) seedlings. One-year-old cork oak seedlings were grown in pots in a factorial experiment with four light treatments (68, 50, 15 and 5% of full sunlight) and two irrigation regimes: well watered (WW) and moderate drought stress (WS). Leaf predawn water potential, which was measured at the end of each of two cycles, did not differ among the light treatments. Water-use efficiency, assessed by carbon isotope composition (delta(13)C), tended to increase with increasing irradiance. The trend was similar in the WW and WS treatments, though with lower delta(13)C in all light treatments in the WW irrigation regime. Specific leaf area increased with decreasing irradiance, and was inversely correlated with delta(13)C. Thus, changes in delta(13)C could be explained in part by light-induced modifications in leaf morphology. The relationship between stomatal conductance to water vapor and net photosynthesis on a leaf area basis confirmed that seedlings in higher irradiances maintained a higher rate of carbon uptake at a particular stomatal conductance, implying that shaded seedlings have a lower water-use efficiency that is unrelated to water availability.     

Effects of root damage associated with Phytophthora cinnamomi on water relations,biomass accumulation,mineral nutrition and vulnerability to water deficit of five oak and chestnut species

The effects of root damage associated with Phytophthora cinnamomi on water relations, biomass accumulation, mineral nutrition and vulnerability to water deficit were investigated in pedunculate oak (Quercus robur), red oak (Quercus rubra) and holm oak (Quercus ilex) saplings over two years. Comparison was made with sweet chestnut (Castanea sativa), a susceptible species to infection by P. cinnamomi, and with a resistant hybrid chestnut (Castanea crenata × C. sativa). Trees were inoculated in 1998 and were subjected to water shortage in 1999. All inoculated sweet chestnuts died before the application of water shortage. Hybrid chestnut, pedunculate oak and red oak displayed low root susceptibility to P. cinnamomi. In these species, water relations, aerial growth and mineral nutrition were slightly affected by inoculation. By contrast, holm oak was the most susceptible oak species to P. cinnamomi as inoculated well‐watered trees displayed the highest root loss (67%) and a 10% mortality. Root loss was associated with a decrease in predawn leaf water potential, a 61% reduction in stomatal conductance, a 55% reduction in aerial biomass, a decrease in leaf carbon isotope discrimination and reduced leaf N and P contents in comparison with controls. In hybrid chestnut and pedunculate oak, water shortage resulted in a similar decrease of predawn leaf water potential, stomatal conductance and aerial biomass in inoculated and non‐inoculated trees. In red and holm oaks, soil volumetric water content of inoculated trees subjected to water shortage remained high. The effects observed in those trees were similar to those of inoculated well‐watered trees and were probably the result of root infection only.     

Occurrence of Diplodia corticola,including new oak host records,in Wisconsin,USA

In the United States, diseased oaks (Quercus species) exhibit tip blight, branch and stem cankers, and dieback often attributed to Diplodia species or related fungi. Emergence of Diplodia corticola as a pathogen of European oaks, and reports of this fungus in the eastern and western United States, prompted re‐examination of strains from Wisconsin. These had been obtained in the late 1990s and early 2000s and previously identified only as Diplodia species. Nuclear ribosomal ITS sequences were obtained from the Wisconsin strains and analysed with other sequences from GenBank. Wisconsin strains confirmed as D. corticola were from northern red oak (Q. rubra), black oak (Q. velutina), white oak (Q. alba) and bur oak (Q. macrocarpa). Other strains from oaks in Wisconsin were D. mutila and D. seriata. Wound inoculation of northern red, white and bur oak seedlings with D. corticola in a greenhouse resulted in shoot death and stem lesions, from which the pathogen was reisolated. We conclude that D. corticola has been present in the northcentral United States for at least two decades and report two previously unrecognized hosts of this pathogen: white oak and bur oak. The roles of D. corticola, related fungi and influences of other environmental factors in deterioration of oak health in North America merit additional investigation.     

Occurrence of Phytophthora cinnamomi in cork oak forests in Italy

An increasing decline and mortality of cork oak trees have been recently observed in central Italy and Sardinia Island. Following surveys conducted in three declining cork oak forests, a Phytophthora species was consistently isolated from soil samples collected from trees displaying different level of decline. Based on morphological features, growth rates at different temperatures and analysis of DNA sequences of the ITS region, all isolates were identified as Phytophthora cinnamomi Rands. This pathogen caused large brownish lesions on inoculated freshly cut branches of cork oak. It was re‐isolated from all infected tissues. These findings represent the first report of P. cinnamomi on cork oak trees in Italy.     

Etiology of oak decline in Spain

In different areas of Extremadura, Western Spain, soil samples were taken at the bottom of holm oak (Quercus ilex) trees that were showing decline symptoms. Half of each sample was sterilized, and acorns were sown in both sterilized and nonsterilized soil samples. The resulting seedlings were used as baits for the isolation of fungi. Seedlings growing on the natural, nonsterilized substrate were characterized by having a lower vegetative growth than the ones growing on the sterilized soil samples, and most of them died. Phytophthora dnnamomi was consistently isolated from their roots. Fusarium oxysporum was also isolated as well as different species of Pythium, although to a lesser extent. Pathogenicity tests were performed on holm oak seedlings with five different isolates of P. chinnamomi, with F. oxysporum, Pythium and with a mixture of the three fungi. All the inoculated seedlings with P. chinnamomi developed root rot and grew slowly, and 35.7% of them died up to the end of the experiments. P. chinnamomi was consistently isolated from their roots, indicating that this fungus is the causal agent of holm oak decline. However, F. oxysporum caused similar symptoms on oak seedlings as P. chinnamomi, and was isolated also from the roots, although its frequency was lower than that of. P. cinnamomi.     

Testing systemic fungicides for control of Phytophthora oak root disease

Potassium phosphite (PP) formulations registered as fertilizers are now prohibited in Spain. Therefore, we evaluated the systemic fungicide fosetyl‐aluminium (fos‐al) in comparison with PP, against root rot caused by Phytophthora cinnamomi in Quercus woodlands. The direct effect of both systemic fungicides was evaluated in vitro on P. cinnamomi mycelial growth. Protection of cork and holm oak against infection was also evaluated in planta. Metalaxyl was included in both in vitro and in planta experiments for comparison purposes. At 100 μg/mL, PP totally inhibited colony radial growth, in comparison with 75% achieved by fos‐al. At doses recommended by manufacturers, with fos‐al and metalaxyl applications, root symptoms remained similar to the uninfected control levels. Based on these results, fos‐al is a candidate substitute product for PP in Quercus woodlands for control of Phytophthora oak root disease.     

Decline of Mediterranean oak trees and its association with Phytophthora cinnamomi: a review

Mortality events in cork and holm oaks have occurred in the Mediterranean basin since the beginning of the XX century, but severity of decline increased during the 1980s. By that time, the exotic soil borne pathogen Phytophthora cinnamomi was often recovered from declining stands and since then it has been considered the main factor associated with decline. This work analyses data concerning P. cinnamomi surveys in cork and holm oaks trees, pathogenicity tests carried out in controlled experiments, studies about the influence of site characteristics in tree decline and approaches to control the disease. Results of field surveys showed that the pathogen is widespread and pathogenicity tests suggested that host susceptibility to the pathogen is moderate when seedlings are in appropriate watering conditions, particularly cork oaks. Occurrence of decline is also associated with soil characteristics that interfere with root expansion and water retention. We assessed the relative importance of each factor involved in decline and revised the role of P. cinnamomi in cork and holm oak decline.     

Evaluation of fungicides for the control of Botryosphaeria corticola on cork oak in Catalonia (NE Spain)

Botryosphaeria canker of cork oak (Quercus suber L.), caused by Botryosphaeria corticola, is a well-known factor contributing to the decline of this forest species and is a serious disease in the main cork-producing countries of the Western Mediterranean basin. In this work, fungicides with the potential for controlling the disease were selected using a combination of in vitro tests and field trials. Fourteen fungicides in different chemical families were tested in vitro to evaluate their effect on inhibition of mycelial growth. The most effective fungicides were benomyl, carbendazim, cyprodinil + fludioxonil, thiabendazol and thiophanate-methyl; these compounds were then further tested under field conditions. In the field trials, carbendazim and thiophanate-methyl were the most effective fungicides, as shown by efficacy indices, in reducing debarked trunk surface affected by cankers (76% and 50–65%, respectively). These results suggest that the occurrence of Botryosphaeria canker on cork oak can be reduced effectively with the application of selected fungicides after cork removal from the trees.     

Phytophthora disease of Quercus ilex in south‐western Spain

Oak decline that was affecting three holm oak sites in the province of Huelva (south‐western Spain) was studied during 1998–1999. The syndromes of dieback and sudden death have been observed and, in both cases, foliar symptoms were associated with root rot. Characterization of the fungal isolates from necrotic roots led us to identify Phytophthora cinnamomi A2 as consistently associated with the disease. The optimum growth temperatures of these isolates were very high (30°C). Inoculation tests under controlled conditions demonstrated the pathogenicity of the isolates on holm and cork oak seedlings. None of the other biotic factors of Mediterranean oak decline that have been previously described were found in the present study and so, in this case, the forest decline model does not seem to be necessary in order to explain the disease observed. The defoliation and mortality of the oaks was primarily caused by P. cinnamomi, although some abiotic factors such as alternating periods of drought and wet weather in the region may play an important role.     

Influence of water-stress acclimation and <Emphasis Type="Italic">Tuber melanosporum</Emphasis> mycorrhization on <Emphasis Type="Italic">Quercus ilex</Emphasis> seedlings

Mycorrhizal and non-mycorrhizal holm oak (Quercus ilex L.) seedlings inoculated with black truffle (Tuber melanosporum) were grown under nursery conditions and subjected to drought hardening for 4 months in autumn and winter followed by irrigation for 10 days. Leaf water potential and stomatal conductance were monitored during the 4 months of drought. When the test was completed (March), measurements were made for each treatment (inoculated or non-inoculated), and watering regime (watered and water-stressed). Pressure–volume curves, osmotic potential at full turgor, osmotic potential at zero turgor and the tissue modulus of elasticity near full turgor were calculated. Mycorrhizal colonization and growth, and the content of the main mineral nutrients N, P, K, Ca and Mg were measured. Water stress affected plant growth, caused an elastic adjustment of the plant tissues, and decreased the P and K content, and inoculation improved the nitrogen content. Drought acclimation apparently achieved the goal of improving the drought tolerance of holm oak seedlings, without depressing ectomycorrhizal root colonization by T. melanosporum. José Antonio Rodríguez Barreal—Deceased     

Competition for soil water between perennial bunch-grass (Elymus glaucus B.B.) and blue oak seedlings (Quercus douglasii H. & A.)

The competition effects of the perennial bunch-grass (Elymus glaucus B.B.) on the growth and survival of the oak seedlings (Quercus douglasii H. & A.) were investigated. There were four levels of Elymus competition, replicated three times. The three densities ofElymus employed were zero (control), 50 (Low — ‘L’ -), 116 (Medium — ‘M’ -) and 199 (High — ‘H’ -) plants m^?2. Rates of soil water depletion, stomatal conductance, transpiration, shoot elongation and leaf expansion rates were measured between 23 March and 26 May 1988. Rates of soil water depletion, stomatal conductance and transpiration differed amongst the treatments and were higher in the control for the duration of the experiment. Shoot elongation rate (SER) and leaf expansion rate (LER) of blue oak seedling were directly related to soil water potentials. Zero values of LER rates for all treatments were observed at soil water potentials lower than?1.91 MPa, and concurrent reductions of stomatal conductance indicated stomatal closure due to the soil water deficit. In the control treatment, transpiration alone was not high enough to deplete soil moisture and to reduce LER of the oak seedlings. Leaf dessication occurred first in the H and M treatments (53% of seedlings dessicated) and two weeks later in the L treatment (37% dessicated) when the soil water potential was approximately ?4.0 MPa. The number of reproductive tillers and seed dry weight indicated thatElymus plants were under water stress from April 25 and concluded on May 25 with an early summer dormancy in all treatments. Data indicated that light intensity of 50% of ambient did not limit the development of oak seedlings. The results suggested that density of the perennial bunch-grassElymus glaucus lower than 50 plants m^?2 could allow survival and successful establishment of blue oak in understories.     

Influence of site factors on the impact of Phytophthora cinnamomi in cork oak stands in Portugal

Although decline of cork (Quercus suber) and holm oak trees (Quercus rotundifolia) has been described in Portugal in the late years of the 19th century, its development has become a motive of high concern during the last two decades. The presence of Phytophthora cinnamomi in cork and holm oak stands was surveyed in four different regions of the country (Trás‐os‐Montes, Alentejo, Ribatejo and Algarve) during 1995–98. Tree decline severity, sudden death and site characteristics were assessed in 56 sites representing varied conditions. The pathogen was isolated from oak roots and rhizosphere samples in 27 of those places. Other plant species from natural vegetation were sampled in three active disease centres. This survey showed that 56% of the surveyed species of shrub flora were infected with P. cinnamomi, which was detected mainly on species belonging to the families Ericaceae, Cistaceae and Leguminosae. Recovery of P. cinnamomi was more frequent in shallow soils (Leptosols and complex Leptosols and Luvisols). These soils are more common in the south (Algarve), where decline has a high impact. Soils with low fertility and low mineral nutrient levels, particularly phosphorus, seemed to favour infection. Site aspect and topographic tree situation were also evaluated. Sites facing south showed higher occurrence of P. cinnamomi, which was also more frequent in slopes and valleys than on hilltops. In Algarve, a relationship could be established between the crown status and the presence of P. cinnamomi in roots and rhizosphere. Different morphotypes of P. cinnamomi could be distinguished in vitro, and their occurrence in the field was correlated with particular site characteristics. Further research needs and management strategies to limit the extension of the disease are discussed.     

Fire-driven oak regeneration in French Mediterranean ecosystems

Regeneration by seeds for cork oak (Quercus suber) and companion oaks (holm oak Quercus ilex and downy oak Quercus pubescens) is likely to be poor in the fire-prone Maures massif (southern France) but the causes are poorly known. Our objective was to assess the effective recruitment for these three oak species and their temporal pattern of recruitment, in order to determine the main limitation factors and the regeneration window of each species. We studied oak recruits (height <3 m) in naturally regenerated populations according to a gradient of fire recurrence and in five main vegetation types including shrublands and mixed mature woodlands. Fire recurrence was the main explanatory factor of oak recruitment, either directly or through vegetation type and microsite characteristics. The results indicate nil to low recruitment for holm oak and downy oak in shrublands, especially those recurrently burned and dominated by Cistus species. Cork oak recruited better than the other oaks in medium and high shrublands dominated by Erica arborea. In contrast, recruitment was high for holm and downy oak in mixed oak stands and mixed pine-oak stands that have not burned for decades. Microsite conditions such as coverage by litter and shrubs influenced oak recruitment, whereas landscape configuration and stand basal area had no influence. Our results suggest that strategic shrub-clearing, oak planting and protection of mixed oak woodlands as seed sources would help maintaining oak populations in the woodland–shrubland mosaic.     

Influence of overstory density on ecophysiology of red oak (Quercus rubra) and sugar maple (Acer saccharum) seedlings in central Ontario shelterwoods

A field experiment was established in a second-growth hardwood forest dominated by red oak (Quercus rubra L.) to examine the effects of shelterwood overstory density on leaf gas exchange and seedling water status of planted red oak, naturally regenerated red oak and sugar maple (Acer saccharum Marsh.) seedlings during the first growing season following harvest. Canopy cover of uncut control stands and moderate and light shelterwoods averaged 97, 80 and 49%, respectively. Understory light and vapor pressure deficit (VPD) strongly influenced gas exchange responses to overstory reduction. Increased irradiance beneath the shelterwoods significantly increased net photosynthesis (P(n)) and leaf conductance to water vapor (G(wv)) of red oak and maple seedlings; however, P(n) and G(wv) of planted and naturally regenerated red oak seedlings were two to three times higher than those of sugar maple seedlings in both partial harvest treatments, due in large part to decreased stomatal limitation of gas exchange in red oak as a result of increased VPD in the shelterwoods. In both species, seedling water status was higher in the partial harvest treatments, as reflected by the higher predawn leaf water potential and seedling water-use efficiency in seedlings in shelterwoods than in uncut stands. Within a treatment, planted and natural red oak seedlings exhibited similar leaf gas exchange rates and water status, indicating little adverse physiological effect of transplanting. We conclude that the use of shelterwoods favors photosynthetic potential of red oak over sugar maple, and should improve red oak regeneration in Ontario.     

Thermal optima of photosynthetic functions and thermostability of photochemistry in cork oak seedlings

Temperature effects on photosynthesis were studied in seedlings of evergreen Mediterranean cork oak (Quercus suber L.). Responses to changes in temperature and the temperature optima of maximal carboxylation rate (V(cmax)) and maximal light-driven electron flux (J(max)) were estimated from gas exchange measurements and a leaf-level photosynthesis model. The estimated temperature optima were approximately 34 and 33 degrees C for V(cmax) and J(max), respectively, which fall within the lower range of temperature optima previously observed in deciduous tree species. The thermostability of the photosynthetic apparatus was estimated according to the temperature at which basal chlorophyll a fluorescence begins to increase (T(c)). The T(c) was highly variable, increasing from 42 to 51 degrees C when ambient temperature rose from 10 to 40 degrees C, and increasing from 44 to 54 degrees C with decreasing soil water availability while net CO(2) assimilation rate dropped to almost zero. When a heat shock was imposed, an additional small increase in T(c) was observed in drought-stressed and control seedlings. Maximal T(c) values following heat shock were about 56 degrees C, which, to our knowledge, are the highest values that have been observed in tree species. In conclusion, the intrinsic temperature responses of cork oak did not differ from those of other species (similar T(c) under ambient temperature and water availability, and relatively low thermal optima for photosynthetic capacity in seedlings grown at cool temperatures). However, the large ability of cork oak to acclimate to drought and elevated temperature may be an important factor in the tolerance of this evergreen Mediterranean species to summer drought and high temperatures.     

Drought tolerance and transplanting performance of holm oak (Quercus ilex) seedlings after drought hardening in the nursery

Drought stress is the main cause of mortality of holm oak (Quercus ilex L.) seedlings in forest plantations. We therefore assessed if drought hardening, applied in the nursery at the end of the growing season, enhanced the drought tolerance and transplanting performance of holm oak seedlings. Seedlings were subjected to three drought hardening intensities (low, moderate and severe) for 2.5 and 3.5 months, and compared with control seedlings. At the end of the hardening period, water relations, gas exchange and morphological attributes were determined, and survival and growth under mesic and xeric transplanting conditions were assessed. Drought hardening increased drought tolerance primarily by affecting physiological traits, with no effect on shoot/root ratio or specific leaf mass. Drought hardening reduced osmotic potential at saturation and at the turgor loss point, stomatal conductance, residual transpiration (RT) and new root growth capacity (RGC), but enhanced cell membrane stability. Among treated seedlings, the largest response occurred in seedlings subjected to moderate hardening. Severe hardening reduced shoot soluble sugar concentration and increased shoot starch concentration. Increasing the duration of hardening had no effect on water relations but reduced shoot mineral and starch concentrations. Variation in cell membrane stability, RT and RGC were negatively related to osmotic adjustment. Despite differences in drought tolerance, no differences in mortality and relative growth rate were observed between hardening treatments when the seedlings were transplanted under either mesic or xeric conditions.