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.     

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.     

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.     

Occurrence of Phytophthora species in oak stands in Italy and their association with declining oak trees

Soil‐borne species of Phytophthora were isolated from 19 of 30 examined oak forest areas in Italy. The frequency of isolated Phytophthora spp. (35.2%) was significantly correlated with soil pH and longitude of the sites. Eleven Phytophthora species were detected. Phytophthora cambivora, P. cinnamomi and P. cactorum were recovered from sites in central and southern Italy whereas P. quercina was isolated in the northern and central part of the country. Phytophthora citricola occurred all over Italy. Phytophthora quercina was the only species significantly associated with declining oak trees.     

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.     

Involvement of Phytophthora species in white oak (Quercus alba) decline in southern Ohio

This study was initiated to investigate the possible role of Phytophthora species in white oak decline (Quercus alba) in southern Ohio at Scioto Trail State Forest. Surveys demonstrated the presence of four species of Phytophthora including one novel species. By far, the most common species was P. cinnamomi; P. citricola and P. cambivora were isolated infrequently. In few instances, P. cinnamomi was isolated from fine roots and necroses on larger roots. No special pattern of incidence was found, but P. cinnamomi was more commonly isolated from greater Integrated Moisture Index values suggesting moist lower bottomlands favour this Phytophthora species. When tree crown condition was examined relative to the presence of Phytophthora, no significant association was found. However, roots of declining P. cinnamomi‐infested trees had 2.5 times less fine roots than non‐infested and healthy trees, which was significantly different. The population densities of P. cinnamomi from declining trees were significantly greater than from healthy trees, suggesting increased pathogen activity that has the potential to cause dieback and decline and possibly the cause of a reduced fine root amount found on declining trees.     

Variation of endophytic cork oak‐associated fungal communities in relation to plant health and water stress

The main objective of this study was to obtain more comprehensive knowledge about the effect of water stress on endophytic fungal communities in asymptomatic and declining cork oak trees. Six asymptomatic and six declining cork oak trees were randomly selected in a natural cork oak forest located in Sardinia, Italy. In February 2003, the soil around three asymptomatic and three declining trees was covered with a circular plastic film to reduce rain water supply with the intention to induce water stress. The remaining six trees served as controls. Predawn xylematic water potential (PWP) was used as water status indicator and measured seasonally. Between July 2003 and June 2004, fungal endophytes were isolated every 2–4 months from twigs, branches and woody tissues. Significant differences in PWP between covered and control trees were detected mainly in autumn. Gas exchange was greatest in asymptomatic control plants. All tissues were colonized by endophytic fungi. Nineteen fungal species were isolated from 1620 plant fragments. Biscogniauxia mediterranea was the most frequently isolated fungus. Its isolation frequency was significantly higher in declining covered trees than in control trees (p < 0.05). Presence of this fungus in asymptomatic control trees was significantly higher in winter than in summer. Water stress seems to reduce species diversity of the endophytic mycobiota in cork oak and to promote proliferation of some potentially pathogenic endophytes.     

The role of yellow lupin (Lupinus luteus) in the decline affecting oak agroforestry ecosystems

Phytophthora cinnamomi is a soilborne pathogen causing root rot in Mediterranean Quercus species growing in ‘dehesa’ rangeland ecosystems. Recently, it has been reported causing wilting and death of Lupinus luteus (yellow lupin), a spontaneous plant in southern Spain rangelands, but also frequently sowed for livestock grazing. In soils artificially infested with P. cinnamomi chlamydospores and planted with different cultivars of yellow lupin, a significant increase in the density of propagules was detected in comparison with the initial levels of inoculum and with the infested but not planted soil (control). In oak‐rangelands in which yellow lupine was planted, isolation and counting of colonies of P. cinnamomi from soil samples have shown the ability of this plant to maintain or even increase the inoculum density and thus facilitate the infection of trees. Results suggested that cultivation of yellow lupin in oak‐rangeland ecosystems should be avoided whether oak trees are affected by root disease caused by P. cinnamomi or not. This leguminous plant can act as an inoculum reservoir or even enhance inoculum soil levels available for oak root infections, exacerbating the oak decline severity in the region.     

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.     

Temporal metabolic profiling of the Quercus suber–Phytophthora cinnamomi system by middle‐infrared spectroscopy

The oomycete Phytophthora cinnamomi is an aggressive plant pathogen, detrimental to many ecosystems including cork oak (Quercus suber) stands, and can inflict great losses in one of the greatest ‘hotspots’ for biodiversity in the world. Here, we applied Fourier transform‐infrared (FT‐IR) spectroscopy combined with chemometrics to disclose the metabolic patterns of cork oak roots and P. cinnamomi mycelium during the early hours of the interaction. As early as 2 h post‐inoculation (hpi), cork oak roots showed altered metabolic patterns with significant variations for regions associated with carbohydrate, glycoconjugate and lipid groups when compared to mock‐inoculated plants. These variations were further extended at 8 hpi. Surprisingly, at 16 hpi, the metabolic changes in inoculated and mock‐inoculated plants were similar, and at 24 hpi, the metabolic patterns of the regions mentioned above were inverted when compared to samples collected at 8 hpi. Principal component analysis of the FT‐IR spectra confirmed that the metabolic patterns of inoculated cork oak roots could be readily distinguished from those of mock‐inoculated plants at 2, 8 and 24 hpi, but not at 16 hpi. FT‐IR spectral analysis from mycelium of P. cinnamomi exposed to cork oak root exudates revealed contrasting variations for regions associated with protein groups at 16 and 24 h post‐exposure (hpe), whereas carbohydrate and glycoconjugate groups varied mainly at 24 hpe. Our results revealed early alterations in the metabolic patterns of the host plant when interacting with the biotrophic pathogen. In addition, the FT‐IR technique can be successfully applied to discriminate infected cork oak plants from mock‐inoculated plants, although these differences were dynamic with time. To a lesser extent, the metabolic patterns of P. cinnamomi were also altered when exposed to cork oak root exudates.     

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.     

Influence of water stress on susceptibility of red oak (Quercus rubra) to Phytophthora cinnamomi

Influence of water status of oak trees (Quercus rubra) on the development of Phytophthora cinnamomi lesions was studied. On agar media or excised inner bark, growth of P. cinnumomi was reduced by low water potentials. In contrast, on voung saplings or mature oak trees the development of P. cinnamomi lesions was not affected during a period of water stress. But after the end of the water stress treatment, susceptibility of the young saplings to P. cinnamoni increased.     

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.     

Cork oak (<Emphasis Type="Italic">Quercus suber</Emphasis> L.) wood growth and vessel characteristics variations in relation to climate and cork harvesting

Variations in tree ring growth of Quercus suber L. were analysed using dendrochronological techniques on cork oak discs from trees harvested in the cork producing region of Alentejo, Portugal. A tree-ring chronology containing a strong common signal and covering the period from 1970 to 1995 was build for ca. 30-year-old cork oaks never submitted to cork harvesting using 14 trees that crossdated satisfactorily out of 30 sampled trees. The tree ring indices correlated positively with September temperature (r = 0.48, P < 0.05) and very strongly with precipitation totals from previous October until current February (r = 0.82, P < 0.001) showing that the water stored in the soil during the autumn and winter months prior to the growing season has a primordial effect on the growth of the given season. The effects of cork harvesting were analysed by comparing mean ring width, mean annual vessel area, vessel density (n°vessels/mm²), and vessel coverage (percentage of transverse surface occupied by vessels) between three mature cork oak trees and three young trees, for the period from 1987 to 1996, corresponding to the growth between two consecutive cork removals in the case of mature trees. In 1988, 1989 and 1996 (corresponding to the first and second years after cork removal, and 1996 to a year of cork removal), the ratios between ring widths of young versus mature trees was twice that for the rest of the period. However, an effect of cork removal indicated by eventual alterations in vessel size and distribution in the wood rings corresponding to the years 1988, 1989 and 1996 in the mature cork oaks was not observed.     

Treatment of oak decline using pressurized injection capsules of antifungal materials

Quercus ilex and Quercus suber trees growing at several sites in Extremadura, Western Spain, that were showing symptoms of oak decline were injected with potassium phosphonate, quinosol or carbendazim using a low-pressure method of trunk injection composed of a pressurized capsule system. A team of four people injected between 120 and 189 trees per day, depending upon the density of the undergrowth vegetation. This labour cost represented, approximately, 15–20% of the total cost of the treatment. The potassium phosphonate-injected trees showed a significant improvement in vegetative growth within 2 years of the injection treatment, and they also showed some recovery from the decline symptoms during the third year. Only one injection treatment of an average of 3.5 capsules (corresponding to 24.5 g phosphonic acid) per tree of approximately 36 cm in diameter, was necessary to reduce the disease severity significantly. Indirectly, these results corroborate the implication of Phytophthora cinnamomi in oak decline within Spanish Quercus woodlands.     

Factors affecting post-fire crown regeneration in cork oak (Quercus suber L.) trees

Cork oak (Quercus suber) forests are acknowledged for their biodiversity and economic (mainly cork production) values. Wildfires are one of the main threats contributing to cork oak decline in the Mediterranean Basin, and one major question that managers face after fire in cork oak stands is whether the burned trees should be coppiced or not. This decision can be based on the degree of expected crown regeneration assessed immediately after fire. In this study we carried out a post-fire assessment of the degree of crown recovery in 858 trees being exploited for cork production in southern Portugal, 1.5 years after a wildfire. Using logistic regression, we modelled good or poor crown recovery probability as a function of tree and stand variables. The main variables influencing the likelihood of good or poor crown regeneration were bark thickness, charring height, aspect and tree diameter. We also developed management models, including simpler but easier to measure variables, which had a lower predictive power but can be used to help managers to identify, immediately after fire, trees that will likely show good crown regeneration, and trees that will likely die or show poor regeneration (and thus, potential candidates for trunk coppicing).     

Fine root dynamics of oak saplings in response to Phytophthora cinnamomi infection under different temperatures and durations

The belowground effects of Phytophthora cinnamomi on 1‐year‐old saplings of two common oak species in mid‐Atlantic US forests, white (Quercus alba) and red oak (Q. rubra), were examined after incubation in pathogen‐infested soilless potting mix. Fine root lengths (0–1.5 mm in diameter) of both oak species were quantified after incubation at successive 30‐day intervals up to 300 days, for a total of 10 incubation periods. In addition, colony‐forming units (CFU) of P. cinnamomi were quantified after white oak saplings were incubated in infested soilless potting mix at different temperature/duration combinations that reflect soil conditions present in the mid‐Atlantic United States. Impact of P. cinnamomi on fine root lengths of red and white oak saplings varied considerably over time. Significant periods of fine root loss occurred primarily during spring, when bud break and leaf flush began for both oak species. Red oaks had 17% fine root loss on average, while white oaks appeared more resistant to P. cinnamomi infection with a 2% decrease in fine roots over the course of the experiment. Phytophthora cinnamomi CFU declined significantly with exposure to all incubation temperatures except 8°C. This was in contrast to in vitro experiments, where the optimum temperature for mycelial growth was determined to be 21°C and above. Significant fine root loss caused by P. cinnamomi depended on plant phenology and the oak species tested. Extreme soil temperatures have a significant adverse impact on temporal changes of P. cinnamomi population.     

Differences in composition of endophytic mycobiota in twigs and leaves of healthy and declining Quercus species in Italy

The composition of the endophytic fungal assemblages in Quercus cerris, Q. pubescens and Q. robur, three oak species most susceptible to decline, was investigated in Fagarè (Padova), Ulignano (Pisa), and Radda in Chianti (Firenze). The endophytic assemblages were studied as a function of health status of the whole tree and of twigs and leaves. The effect of station altitude was tested on Q. cerris. Samples were gathered from healthy and declining trees. In April (bud opening time) 20 twigs and 10 leaves were collected from each tree. Fungi were isolated from twig segments and leaf portions. A total of 23 fungal genera with 27 different species were isolated, in addition to a few sterile mycelia. Eleven species were found to be common to all three oak species, while other species were found only on one oak species, suggesting host specificity. Among the fungal species isolated, some were known to be pathogenic (Apiognomonia quercina, Colpoma quercinum, Diplodia mutila, Phomopsis quercina). The colonization frequency (CF) of pathogenic species varied between 0.9% for A. quercina in Q. cerris and 60.2% for P. quercina in Q. robur. Analysis of variance showed a statistically significant difference among the oak species tested. The CF was higher in declining trees in comparison with healthy trees, and also in twigs vs. leaves. The CF was found to be higher for Q. cerris trees growing at sea level as compared with those situated at an elevation of 350–400 m. In stands where, on account of particular ecological conditions, pathogenic behaviour is displayed simultaneously by more than one endophytic fungal species, trees accentuate their decline.     

Susceptibility of the Quercus rubra root system to Phytophthora cinnamomi; comparison with chestnut and other oak species

The susceptibility of oak seedlings (Quercus palustris, Quercus robur, Quercus rubra) and chestnut seedlings (Castanea sativa) to Phytophthora cinnamomi was tested. The dynamics of infection was examined in plant material raised in a rhizotron. In the oak species, primary root tissues were susceptible whereas secondary cortical tissues showed some resistance to P. cinnamomi. Secondary cortical tissues of the tap root in C. sativa were susceptible. Inoculations with P. cinnamomi were performed both in situ and on excised roots of mature Q. rubra. In both cases, the resistance of Q. rubra roots and shoots was negatively correlated with diameter at the inoculation point. Small roots (l–5-cm diameter) were resistant, whereas collar and trunk were susceptible. In contrast to oak, small excised roots of mature C. sativa (0.7–2-cm diameter) were susceptible to P. cinnamomi. This may explain why P. cinnamomi does not induce a decline of the attacked oaks, but rather a trunk canker.     

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.