Weed control modifies Tuber melanosporum mycelial expansion in young oak plantations

Context

Black truffle (Tuber melanosporum) cultivation is a promising agro-forestry alternative for Mediterranean rural areas, but adequate weed control at seedling establishment still remains a challenge in black truffle plantations.

Aims

The aim of this study is to evaluate the effects of several weed control strategies on early development of Quercus ilex seedlings and the symbiotic T. melanosporum.

Materials and methods

In a young black truffle-inoculated holm oak plantation, we assessed for 3 years the effects of two types of mechanical weed control and five mulches in a young Q. ilex plantation inoculated with T. melanosporum. Herbaceous cover, seedling growth and abundance of T. melanosporum mycelium, based on PCR analysis of soil DNA extracts using T. melanosporum-specific primers, were estimated to determine the effectiveness of these treatments in controlling weeds and supporting the growth of both the host tree and the target fungus.

Results

The amount of T. melanosporum mycelium in the soil 30 cm around the seedlings was larger under double-layer white mulch than in the rest of treatments tested. Under the white colour mulches, which had the largest light reflection, we registered the cooler soil temperature, and the best weed control was observed on the single- and double-layer black truffles and double-layer white mulch.

Conclusion

The effects of double-layer white mulch on herbaceous cover, soil temperature, reflected light, and the expansion of T. melanosporum bring us closer to being able to substitute traditional tilling of truffle orchards for the less expensive mulching treatments.     

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     

Pathogenicity of three Phytophthora spp. causing late seedling rot of Quercus ilex ssp. ballota

Within a research project on quality of plants for forestation of agricultural lands, we studied the aetiology of a late seedling rot affecting holm oak (Quercus ilex ssp. ballota) in two forest nurseries in southern Spain. Major disease symptoms were foliage wilting and necrosis of feeder roots. Phytophthora cinnamomi, Phytophthora cryptogea and Phytophthora drechsleri were isolated from necrotic roots of holm oaks. Selected isolates of the three Phytophthora species were pathogenic to Quercus ilex ssp. ballota and Quercus suber seedlings in artificial inoculations. Soil flooding conditions were essential for infection and root rot development. There was no host specificity among the species, the isolates of P. cinnamomi being the most virulent in all inoculated plants. In these inoculations, Q. ilex ssp. ballota plants were more susceptible than those of Q. suber. This work is the first report of P. cinnamomi, P. drechsleri and P. cryptogea affecting Q. ilex ssp. ballota in forest nurseries.     

Monitoring the fate of a 30-year-old truffle orchard in Burgundy: from Tuber melanosporum to Tuber aestivum

Truffles, i.e. tree root-associated fungal fruiting bodies, clearly range among the world’s most exclusive delicacies. Despite the quite restricted natural geographic occurrence of one of the most renowned fungal species, namely Tuber melanosporum, the development of inoculation procedures in the late 1960s made it possible to enlarge its production area in different countries. This was achieved by planting orchards with host tree seedlings colonized by the fungus. In the present work, we investigate the behavior of one of the earliest T. melanosporum orchards planted in Burgundy (France) over a long-term scale (more than 30 years). A picture of the orchard evolution was obtained by recording truffle yields and fungal morphotypes over the seasons and relating them to host-tree development and climate data. The most relevant results include the time-delayed, but rather fast replacement of inoculated T. melanosporum by naturally occurring T. aestivum and the key role of climate in the inter-annual variability of truffle production.     

Weed management and irrigation are key treatments in emerging black truffle (Tuber melanosporum) cultivation

Interest in conversion of marginal agricultural lands to small oak woodlands for the production of black truffles (Tuber melanosporum Vittad.) is increasing in the native black truffle areas of France, Spain and Italy as well as suitable or amended sites throughout the world due to high economic returns for gastronomically valuable truffles. Successful long-term management of a perennial plantation replacing an annual crop requires understanding the system in all phases of the life cycle, and in the case of truffles this involves understanding the interactive growth of the aboveground host and the belowground symbiotic ectomycorrhizal fungus whose fruitbody is the desired truffle. Here we focus on the pre-production phase, 4 years after establishing the truffle-oak plantation and prior to truffle production. We tested the influence of weed control, irrigation and fertilizer, each at 3 levels, on plant growth and ectomycorrhizal proliferation in three truffle-oaks plantations in northeast Spain. Results show that adequate weed control improves root and shoot dry weight while fertilizer and irrigation treatments did not influence plant growth. The low dose of irrigation (50% of the estimated water deficit for the site) and glyphosate weed control both increased total root tips/plant and T. melanosporum colonized tips (ectomycorrhizae) by approximately two-fold, compared to control treatments. Distribution of the ectomycorrhizae within the soil profile was significantly influenced by the low dose irrigation treatment, with increases observed in the 10?C20 cm and the 20?C30 cm deep layers compared to the control and high irrigation treatments. Four years after planting, T. melanosporum remained the dominant fungal symbiont, despite the presence of 14 other ectomycorrhizal morphotypes from these sites.     

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.     

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.     

Problems of using pines in <Emphasis Type="Italic">Tuber melanosporum</Emphasis> culture: soils and truffle harvest associated with <Emphasis Type="Italic">Pinus nigra</Emphasis> and <Emphasis Type="Italic">P. sylvestris</Emphasis>

In Mediterranean pine forests, truffles and mushrooms generate greater profits than any other woodland products. However, there are no studies on Tuber melanosporum Vittad. associated with pines. For this reason, we have carried out a study of this truffle in mountain woods with Pinus sylvestris L. and P. nigra Arnold subsp. salzmannii (Dunal) Franco, in central Spain. Two hundred and eight Tuber melanosporum burns were monitored for 7 years in five different habitats within the same geographical area. An ANOVA test confirmed significant differences in carpophore production. In higher producing habitats, pines were less abundant. We also confirmed that in 433 burns, T. melanosporum was always unequivocally associated with the root base of Quercus or Corylus trees. Similarly, 14 truffle collectors confirmed that they had never found a single burn with carpophore production associated exclusively with pines. Nevertheless, soil analyses indicated that the soil of these pine woods was very favourable to Tuber melanosporum. We therefore conclude that at present Pinus nigra salzmannii and P. sylvestris are of little interest to Tuber melanosporum culture, as they hinder carpophore production. However, this study has also confirmed that Pinus nigra salzmannii and P. sylvestris mycorrhize easily with Tuber melanosporum, both in the laboratory and in natural environments. On this basis, we propose that pines may act as transmitters of T. melanosporum, although they do not induce fruiting. As a result, the commercial cultivation of Pinus nigra salzmannii and P. sylvestris seedlings mycorrhized with Tuber melanosporum is not recommended in truffle culture at the present time.     

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.     

Problems of using rockroses in <Emphasis Type="Italic">Tuber melanosporum</Emphasis> culture: soil and truffle harvest associated with <Emphasis Type="Italic">Cistus laurifolius</Emphasis>

The Périgord black truffle (Tuber melanosporum Vittad.) can mycorrhize with rockroses (Cistus L.) in Mediterranean forests. These shrubs may be of interest for truffle cultivation as they produce early carpophores, collaborate in tree mycorrhization and in the survival of Tuber melanosporum mycelium. However, there are very few quantitative studies on truffle production associated with Cistus. For this reason, we have carried out a study on the natural production of Tuber melanosporum associated with Cistus laurifolius L. in central Spain. An Anova test confirmed the existence of significant differences in carpophore production in four habitats, with and without the presence of C. laurifolius. Twenty-one burns associated with C. laurifolius showed less significant production than the rest. We also confirmed that in 122 burns, Tuber melanosporum was always unequivocally associated with the root base of Quercus L. or Corylus L. trees. Similarly, five truffle-collectors confirmed that they had never found a single burn with carpophore production associated exclusively with rockroses. It has also been statistically verified that many Cistus die in Tuber melanosporum burns, whereas in old burns with minimum carpophore production, a new colonization of young Cistus has been observed. This study also confirms that C. laurifolius mycorrhizes easily with Tuber melanosporum. We propose that Cistus laurifolius may act as transmitters of Tuber melanosporum, although they do not induce fruiting. Consequently, the commercial cultivation of Cistus laurifolius mycorrhized with Tuber melanosporum for production is not recommended in truffle culture. The results reported substantiate the negative observations in cultivations of T. melanosporum with other Cistus species.     

Colonization of Pedunculate oak by the Burgundy truffle fungus is greater with natural than with pelletized lime

European black truffles can be profitable agroforestry crops outside their native ranges. Truffle fungi grow symbiotically as ectomycorrhizae on the roots of host trees, notably hazels and oaks. Conditions in the central USA appear conducive to cultivation of the Burgundy truffle (Tuber aestivum Vitt. syn. T. uncinatum Chatin), but research is needed to determine effects of management practices on truffle establishment and fruiting. In a greenhouse study we tested the effect of lime type, inoculation technique, and two truffle sources on Pedunculate oak (Quercus robur L.) growth and mycorrhizal colonization. We found that the type of lime used to raise potting mix pH can differentially affect the growth rate of root systems inoculated with different selections of Burgundy truffle inoculum. Seedlings inoculated with one selection of the truffle and grown in potting mixes amended with natural crushed dolomitic limestone developed larger root systems with more truffle mycorrhizae compared with potting mix amended with high-calcium pelletized quick-release lime. Seedlings inoculated with a second truffle selection were not affected by lime source and developed root systems as large as those developed with the first truffle source grown with natural lime. Supplemental root dip inoculation did not improve levels of colonization beyond those accomplished by potting mix infestation with truffle ascospores. Use of a hygroscopic polymer to maintain ascospore suspension in the inoculum slurry used to infest the potting mix had no effect on root system development or mycorrhiza formation.     

Why do large,nitrogen rich seedlings better resist stressful transplanting conditions? A physiological analysis in two functionally contrasting Mediterranean forest species

We analysed the physiological bases that explain why large and high nitrogen (N) concentration seedlings frequently have improved survival and growth relative to small seedlings in Mediterranean woodland plantations. Large seedlings of Aleppo pine (Pinus halepensis Mill.) and holm oak (Quercus ilex L.) with high N concentration (L+), and small seedlings with either high (S+) or low (S−) N concentration, were planted on two sites of different weed competition intensity that created contrasting stress conditions. Seedling survival, growth, gas exchange, N remobilization (N_R) and uptake (N_U), and water potential were assessed through the first growing season. Weeds reduced survival and growth, but seedling response to weed competition varied among phenotypes and between species. At the end of the first growing season, L+ Aleppo pine seedlings had higher survival than both small seedling types in presence of weeds but no differences were observed in absence of weeds. Mortality differences among phenotypes occurred in spring but not in summer. L+ Aleppo pines grew more than small Aleppo pines independently of weed competition. No holm oak seedling type survived in presence of weeds and no mortality differences among phenotypes where observed in absence of weeds, although L+ holm oak seedlings grew more than small seedlings. Mortality and growth differences in Aleppo pine were linked to marked physiological differences among phenotypes while physiological differences were small among holm oak phenotypes. L+ Aleppo pines had greater root growth, gas exchange, N_R, and N_U than small seedlings, irrespective of their N concentration. Seedling size in Aleppo pine had a greater role in the performance of transplanted seedlings than N concentration. The functional differences among oak phenotypes were small whereas they were large in pine seedlings, which led to smaller differences in transplanting performance in holm oak than in pine. This suggests that the nursery seedling quality improvement for planting in dry sites could depend on the species-specific phenotypic plasticity and functional strategy. Improved transplanting performance in large Aleppo pine seedlings relative to small seedlings was linked to greater gas exchange, root growth and N cycling.     

Introduced <Emphasis Type="Italic">Tuber aestivum</Emphasis> replacing introduced <Emphasis Type="Italic">Tuber melanosporum</Emphasis>: a case study

A Tuber melanosporum plantation established in 1994/1995 on Kibbutz Bar’am (in the Upper Galilee, Israel) gradually lost its T. melanosporum mycorrhiza. In 1999, T. aestivum inoculated seedlings were inadvertently introduced into the plantation to fill the gaps between trees. A single T. melanosporum fruit body was found in 1999. Although no truffles were found after 1999 and until 2009, in that year and in 2010, truffles were collected. Morphological and molecular analyses proved these to be T. aestivum. Thus, the intentionally introduced T. melanosporum mycorrhiza was replaced by that of another introduced mycorrhizal fungus, T. aestivum. Local oak species produced higher yields compared to introduced host species known to be good T. melanosporum plant symbionts. The yield was comparable to that reported for young commercial orchards, but the fruiting season was earlier than in Europe.     

Two provenances of Quercus ilex ssp. ballota (Desf) Samp. nursery seedlings have different response to frost tolerance and autumn fertilization

Despite evidences that Holm oak has a high plasticity and great adaptability, there is limited or contradictory knowledge of the morphological and physiological variability of this species. Holm oak has been widely used for reforestation projects in Mediterranean areas, but has frequently shown poor field performance. We hypothesized that Holm oak has inter-population differences in physiological responses to abiotic stressors that could affect reforestation success. The influence of nursery culture on the characteristics of Holm oaks from different provenances has not been explored in depth. Thus, we studied the effect of nursery autumn fertilization on morphological traits, frost tolerance, root growth potential, and nutritional status of two Spanish provenances of Holm oak, La Alcarria (a region with inland Mediterranean climate) and Sierra Morena Occidental (a region with a warm coastal Mediterranean climate). There were significant differences between the provenances in frost tolerance, biomass allocation, and leaf nutrient content, suggesting a role of genetic factors. The leaves of seedlings from La Alcarria had less visual damage at ?12°C than seedlings from the warmer provenance (45% vs. 92%). Seedlings from La Alcarria, compared to those from Sierra Morena, had higher leaf P concentration (0.17% vs. 0.15%), greater stem diameter (3.1?mm vs. 2.7?mm), lower shoot-to-root dry mass ratio (0.46 vs. 0.53), and lower slenderness (4.03 vs. 5.31). For both provenances, N autumn fertilization improved growth, root growth potential, cold hardiness, and nutritional status of seedlings. We suggest that forest reforestation programs should consider to a greater extent Holm oak provenances and their tolerances to different abiotic stressors.     

Growth response, phosphorus content and root colonization of Polylepis australis Bitt. seedlings inoculated with different soil types

Polylepis forests are one of the most endangered high mountain ecosystems of South America and reforestation with native Polylepis species has been highly recommended. Greenhouse bioassays were set up to determine the influence of three different soils on growth and phosphorous nutrition of Polylepis australis seedlings. Soils were collected from a grassland, a rare mature forest and a forest degraded due to repeated fires. We identified the arbuscular mycorrhizal fungi (AMF) present in the three soils and after 12 months we harvested the seedlings to evaluate root and shoot biomass, plant P content and root colonization by native AMF and dark septate endophytes (DSE). The soil inocula contained 26 AMF morphospecies. Grassland inoculum showed the highest AMF richness, and mature forest showed a different AMF community assembly from grassland and degraded forest inocula. Root biomass and root colonization were highest in seedlings inoculated with mature forest soil, meanwhile shoot biomass and plant P content were similar between all treatments. AMF colonization correlated negatively with DSE and root biomass was negatively correlated with DSE colonization, thus these fungal symbionts could be competing for resources. Our results indicate that AMF inoculum from the mature forest stand has the potential to improve P. australis performance, probably due to the dominance of Glomeraceae and Acaulosporaceae families. However, other soil microorganisms could be together with AMF in the natural inocula, affecting the growth response of P. australis seedlings. Future studies evaluating the effect of these inocula under field conditions should be carried out.     

Thinning effects on carbon allocation to fine roots in a Quercus ilex forest

The dynamics of the fine root system are relevant to calculations of the carbon balance of the ecosystem, and there is also a need to quantify changes in this component caused by disturbances. Mediterranean forest systems have historically been coppiced to obtain charcoal. As a result of the resprouting capacity of holm oaks (Quercus ilex L.), these forests present more than 50% of their biomass below ground (stump + roots > 1 cm in diameter), but the effects of thinning on the fine root system are unknown. Fine root biomass, production, mortality and longevity were studied in a control and a thinned Mediterranean holm oak forest by minirhizotron methodology. Observations of fine roots started 2 years after thinning and continued for almost 3 years. Extraction of 80% of the former basal area of the forest greatly affected carbon allocation patterns. Biomass increased by more than 100%, production increased by 76%, mortality increased by 32% and longevity decreased by around 2 weeks. The greatest differences between treatments were associated with differences in growth during autumn months in the top 40 cm of soil, particularly between 10 and 20 cm depth.     

Trends in evergreen oak suitability from assembled species distribution models: assessing climate change in south-western Europe

The expected changes to the environmental conditions have concerned the scientific community over the last few decades. A rise in the mean temperature and a variation in rainfall patterns could modify the current distribution of plant species. In this study, we analysed four evergreen oaks (Quercus ilex subsp. ilex, Q. ilex subsp. ballota, Q. suber and Q. coccifera) by means of species distribution models. Three algorithms were used: maximum entropy, logistic regression and environmental distance. Taxa occurrences were taken, chiefly from the National Forest Inventories, and climate data was retrieved from the WorldClim 1.4 project. The present period and four future scenarios were studied. The latter were carried out by averaging thirteen global circulation models (GCMs). Area under the curve was used for validating the models. Maps indicating the suitability and cosuitability among the evergreen oaks were developed. The potential distribution of evergreen oaks in the present period was found to be wider than the actual distribution. Simulations indicate that climate change would increase the cosuitability of western temperate areas for Mediterranean oaks. The use of different algorithms and GCMs, as well as the high validation values obtained, make the study robust. Oaks are an important source of income, especially Q. ilex subsp. ballota and Q. suber. Our findings contribute to our understanding of the dynamics of oaks, and can be considered for management programmes aimed at conserving this natural heritage.     

Seasonal and spatial mortality patterns of holm oak seedlings in a reforested soil infected with Phytophthora cinnamomi

The viability of 1-year-old holm oak (Quercus ilex) seedlings in a soil naturally infected with Phytophthora cinnamomi was studied during 2 consecutive years in a plot located in south-western Spain. In both years, total mortality during autumn and winter was not noticeable (<2.1%). In spring, mortality levels were higher (8.3–4.6%), especially the first year. A steep increase in total mortality occurred in summer, both in the first (11.4%) and second (24.2%) year, but mortality attributable to P. cinnamomi was 1.9 and 7.6%, respectively. Thus, 2 years after planting, total cumulative mortality was 43.4%, and that attributable to P. cinnamomi 9.6% (i.e. 22.1% of total mortality). Fungus-derived mortality followed a spatially aggregated pattern in the reforestation plot, suggesting a clustered distribution of the inoculum in the soil. Furthermore, mortality by P. cinnamomi was also associated with nearness of infected adult trees in the plot. Results obtained are discussed in the framework of seasonal water deficit, P. cinnamomi damage, weed competition and sanitation techniques to be used in declined holm oak stands in Spain.     

Enrichment planting of native species for biodiversity conservation in a logged tree plantation in Vietnam

Decades of deforestation and over logging have created large expanses of degraded lands in many countries including Vietnam. Reforestation may offer one means of mitigating these processes of degradation while sustaining biodiversity conservation. However a lack of information regarding trees, in particular threatened tree species has been identified as an important limitation in being able to reforest for biodiversity conservation. In the current study, conducted in the Tan Phu “protection forest”, the investigation surrounds the feasibility of biodiversity restoration in a fast-growing plantation after logging. Twelve threatened and native species mainly belonging to the Dipterocarpaceae and Fabaceae families have been considered. Seedlings grown in a local nursery have been planted under the canopy of a 50 ha pioneer forest, with a distance of 5 m between plants and between rows. The experimental design consists of blocks, each one with a replication of a 16-tree plot for each species. Four years after plantation, the survival rate of seedlings is high (>70 %), except for Dipterocarpus costatus (<10 %). Growth shows possible inhibitions under a closed canopy but the mean annual increment is generally satisfactory. The early performance of the seedlings is promising to make this plantation a good example of biodiversity restoration. These results could enhance the reforestation efforts of forest managers and encourage them to use native tree species including threatened ones in order to restore a high level of biodiversity in tropical degraded forests.     

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.