Pinea Seedlings Research Articles

Physiological, metabolic and hormonal responses of two Pinus spp. with contrasting susceptibility to brown-spot needle blight disease.

Needle blights are serious fungal diseases affecting European natural and planted pine forests. Brown-spot needle blight (BSNB) disease, caused by the fungus Lecanosticta acicola, causes canopy defoliation and severe productivity losses, with consequences depending on host susceptibility. To gain new insights into BSNB plant-pathogen interactions, constitutive and pathogen-induced traits were assessed in two host species with differential disease susceptibility. Six-month-old Pinus radiata D. Don (susceptible) and Pinus pinea L. (more resistant) seedlings were needle inoculated with L. acicola under controlled conditions. Eighty days after inoculation, healthy-looking needles from symptomatic plants were assessed for physiological parameters and sampled for biochemical analysis. Disease progression, plant growth, leaf gas-exchanges and biochemical parameters were complemented with hormonal and untargeted primary metabolism analysis and integrated for a holistic analysis. Constitutive differences between pine species were observed. Pinus pinea presented higher stomatal conductance and transpiration rate and higher amino and organic acids, abscisic acid as well as putrescine content than P. radiata. Symptoms from BSNB disease were observed in 54.54% of P. radiata and 45.45% of P. pinea seedlings, being more pronounced and generalized in P. radiata. For both species, plant height, sub-stomatal CO2 concentration and water-use efficiency were impacted by infection. In P. radiata, total soluble sugars, starch and total flavonoids content increased after infection. No differences in hormone content after infection were observed. However, secondary metabolism was induced in P. pinea visible through total phenolics, flavonoids and putrescine accumulation. Overall, the observed results suggest that P. pinea constitutive and induced traits may function as two layers of a defence strategy which contributed to an increased BSNB resistance in comparison with P. radiata. This is the first integrative study linking plant physiological and molecular traits in Pinus-Lecanosticta acicola pathosystem, contributing to a better understanding of the underlying resistance mechanisms to BSNB disease in pines.

Characterization and pathogenicity of Diplodia seriata causing branch canker on Pinus pinea in Tunisia

Several species of Botryosphaeriaceae family are among the most aggressive pathogens associated with botryosphaeria dieback of agricultural and forestry trees. Particularly, Diplodia spp. having a cosmopolitan distribution, are well-known as virulent of woody plant hosts including Pinus spp. In recent years, symptoms of canopy wilt, branch dieback, necrosis, and trunk cankers have been noticed on Pinus pinea trees in Tunisian forests. However, this has been less welldocumented in North Africa and especially in Tunisia. The aim of this study is to characterize the causal agent of P. pinea dieback in northeastern Tunisian forest. A collection of thirty-eight isolates obtained from symptomatic branches of P. pinea trees was identified as Diplodia seriata by means of morphological characteristics, and phylogenetic analysis of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA. A pathogenicity test was conducted on 3-years-old P. pinea seedlings, confirmed the virulence of the fungus. To the best of our knowledge, this is the first record of D. seriata associated with branch canker on P. pinea in Tunisia.

First report of damping off disease caused by Fusarium oxysporum on Pinus pinea in Jordan.

First report of damping off disease caused by Fusarium oxysporum on Pinus pinea in Jordan.

Diplodia Species Causing Dieback on Pinus Pinea: Relationship Between Disease Incidence, Dendrometric and Ecological Parameters

ABSTRACT Forest decline is a widespread phenomenon on coasts throughout the Mediterranean basin. Pathogenic fungi are amongst the main causes of forest tree diseases. Diplodia species, having a cosmopolitan distribution, are well-known as pathogens of woody plant hosts including Pinus spp. In recent years, symptoms of shoot dieback, necrosis, twig blight, canopy transparency and trunk cankers have been observed on Pinus pinea trees in Tunisian forests. Nevertheless, this has been less well-studied in North Africa and especially in Tunisia. The purpose of this study is to report the occurrence of Diplodia species causing dieback on P. pinea in two forests in northeastern and the northern Tunisia. A collection of fifty-eight isolates were obtained from symptomatic branches of P. pinea trees. Three Diplodia species were identified: D. africana, D. pseudoseriata and D. scrobiculata by means of morphological characteristics, and phylogenetic analysis. Their incidence on P. pinea trees appeared to be significantly correlated to the dendrometric parameters and ecological factors. A pathogenicity test was conducted on 3-years-old P. pinea seedlings, confirmed the virulence of three Diplodia species. Based on the length of vascular necrosis in the wood of the P. pinea inoculated stem, D. scrobiculata was the most virulent species. Data from the present study provide the first report of the species of Diplodia associated with P. pinea dieback in Tunisia.

Characterization of Tuber indicum (Pezizales, Tuberaceae) mycorrhizae synthesized with four host trees exotic to China

The Chinese black truffle Tuber indicum is the main commercial truffle species in China and has strong potential for cultivation. Most studies have focused so far on the production and planting of mycorrhizal seedlings of tree species native to China. Here, we selected four exotic tree species, Quercus pubescens, Q. ilex, Q. palustris and Pinus pinea and inoculated axenically germinated seedlings, five replicates per tree species, with T. indicum spore suspension. As shown by morphological, anatomical and molecular analyses, mycorrhizae were successfully synthesized under greenhouse conditions from 6 months after inoculation and the mycorrhization was stable for at least 24 months in the glasshouse environment. Despite slight morphological variations, T. indicum mycorrhizae were similar on all tree species, i.e. swollen, red-brownish with long hyaline emanating hyphae showing right-angle ramifications. Our observations confirmed the similarity of T. indicum mycorrhizae with those of T. melanosporum regardless of the geographic origin of host trees. Four out of five T. indicum-inoculated pine seedlings were cross-contaminated by T. borchii that was inoculated to another group of P. pinea seedlings raised in the same glasshouse. This is the first study to document the mycorrhization of exotic tree species by T. indicum in China and the first report for Q. palustris. With two successful out of five inoculated seedlings, Q. palustris was less receptive to T. indicum mycorrhization than the other two oak species. Further work is needed to assess whether the T. indicum symbioses obtained here are maintained after planting in the field and if ascomata can be produced by exotic tree species under Chinese environmental conditions.

Regeneration dynamics of mixed stands of Pinus pinaster Ait. and Pinus pinea L. in Central Spain

The dynamics of mixed stands are more complex and less studied than those of monospecific stands. The objective of this work was to analyze the variables involved in seedling occurrence and seedling survival in mixed stands of Pinus pinaster and P. pinea in Mediterranean areas. From 2011 to 2016, regeneration of both species was monitored at two sites located in Central Spain. We installed 72 regeneration plots where seedling dynamics were monitored. All the trees in the study areas were measured and mapped. Additionally, we took hemispherical photographs in each regeneration plot. The average density of P. pinea seedlings over the study period was almost 20 times larger than that of P. pinaster. Our results indicate that the seedlings of both species grow under moderate light conditions. In addition, we found that the occurrence of seedlings of both species was related to the structure of the stand. P. pinea seedlings grew where the density and size of P. pinaster trees were low and where P. pinea trees provided moderately sheltered conditions, whereas the number of P. pinaster seedlings was related to under intermediate densities of P. pinaster trees. Furthermore, seedling survival was positively associated with age of the seedlings and negatively with the August average maximum temperature. The temporal continuity of mixed stands of P. pinea and P. pinaster in the study area is compromised by the observed lack of regeneration of P. pinaster.

Responses of Pinus pinea seedlings to moderate drought and shade: is the provenance a differential factor?

The widespread Mediterranean Pinus pinea showed exceptionally low genetic diversity and low differentiation between traits in the adult phase. We explored the adaptation potential of seedlings from four main Iberian provenances during their regeneration phase. We assessed the variability of shoot growth, allometry, physiological traits, and phenotypic plasticity to the interactive effect of light and water environments during 8-month moderate water-stress cycle and after one-week heat wave. The effect of shade and drought was mainly orthogonal whatever the provenance. The inland La Mancha provenance showed higher shoot growth and biomass compared to the southern coastal Depresion-del-Guadalquivir provenance. Following the heat wave, La Mancha presented higher net photosynthetic rates, a lower decrease in maximal quantum efficiency of PSII, and a higher accumulated relative height growth, thus, showing an adaptive advantage. The observed differences corroborated the ecological grouping of the provenances along latitudinal and inland–coastal gradients. We confirmed the high adaptive plasticity of Pinus pinea to the unpredictable Mediterranean environment.

Morphological, physiological and biochemical responses to drought stress of Stone pine (Pinus pinea L.) seedlings

In this study, the effect of irrigation intervals (drought stress) on growth, predawn xylem water potential (Ψ w), the osmotic potential at full turgor (Ψπ 100), the osmotic potential at the turgor loss point (Ψπ TLP), osmotic adjustment and osmotic solutes (soluble sugars and proline) of Pinus pinea L. seedlings were examined. An experiment was carried out under greenhouse conditions using four watering treatments (control, 7-, 14- and 21-day irrigation intervals) in the first growth season; from mid-July to early November. Results showed that irrigation interval had significant effect on growth characteristics, Ψ w, water relation parameters, and osmotic solutes. The increasing irrigation interval significantly decreased the seedling height, root collar diameter, root, stem and needle dry weight, number of lateral branches, root percentage, root:shoot ratio and diameter:height ratio. Ψ w and total soluble sugars decreased while proline content increased with the increase of drought stress. The Ψπ 100 and Ψπ TLP significantly decreased in drought-stressed seedlings compared to control (no stress) seedlings. The results suggest that the impact of drought stress increased with the increase of irrigation interval. Therefore, in the drought-stressed P. pinea seedlings were indicated osmotic adjustment by increasing the proline content and decreasing Ψπ 100 and Ψπ TLP during drought stress. Growth decreased under drought stress conditions in P. pinea seedlings.

Defining the optimal regeneration niche for Pinus pinea L. through physiology-based models for seedling survival and carbon assimilation

Seedling survival in Pinus pinea is controlled by both water status and photosynthetic performance. Optimal regeneration niche for the species is found at mid-shaded sites. Summer survival has been identified as the main bottleneck preventing natural regeneration in Mediterranean forests, although the physiological processes resulting in seedling mortality are not sufficiently known. In the present work, the effect of water status and photosynthetic performance on seedling survival and regeneration niche in a Mediterranean pine (Pinus pinea L.) was analyzed by means of a modeling approach. Midday water potential was modeled as a nonlinear function of environmental factors, and this model was coupled with an existing model for carbon assimilation. A model for seedling survival was then constructed using lifetime analysis techniques, including predicted values of daily and cumulative net assimilation and probability for critical midday water potentials as predictors. The model was applied over a wide range of irradiance environments in order to identify the optimal regeneration niche for the species. Results indicate that midday water potential for P. Pinea seedlings is affected by relative soil water content, leaf temperature, and irradiance, with younger seedlings being more likely to reach critical values. Seedling survival in P. pinea is controlled by both water status and photosynthetic performance, with mortality being triggered by the joint occurrence of low water potentials and negative assimilation rates, although photoassimilates stored during the spring season increase survival in older seedlings. Simulations indicated that seedling survival is optimized in shaded environments, while carbon assimilation reaches maximum values on more open sites, thus the optimal regeneration niche for the species is found on mid-shaded locations.

Early physiological responses of Pinus pinea L. seedlings infected by Heterobasidion sp.pl. in an ozone-enriched atmospheric environment.

The presence of the American root-rot disease fungus Heterobasidion irregulare Garbel. & Otrosina was detected in Italian coastal pine forests (Pinus pinea L.) in addition to the common native species Heterobasidion annosum (Fries) Brefeld. High levels of tropospheric ozone (O3) as an atmospheric pollutant are usually experienced in Mediterranean pine forests. To explore the effect of interaction between the two Heterobasidion species and ozone pollution on P. pinea, an open-top chamber (OTC) experiment was carried out. Five-year-old P. pinea seedlings were inoculated with the fungal species considered (H. irregulare, H. annosum and mock-inoculation as control), and then exposed in charcoal-filtered open-top chambers (CF-OTC) and non-filtered ozone-enriched chambers (NF+) from July to the first week of August 2010 at the experimental facilities of Curno (North Italy). Fungal inoculation effects in an ozone-enriched environment were assessed as: (i) the length of the inoculation lesion; (ii) chlorophyll a fluorescence (ChlF) responses; and (iii) analysis of resin terpenes. Results showed no differences on lesion length between fungal and ozone treatments, whereas the short-term effects of the two stress factors on ChlF indicate an increased photosynthetic efficiency, thus suggesting the triggering of compensation/repair processes. The total amount of resin terpenes is enhanced by fungal infection of both species, but depressed by ozone to the levels observed in mock-inoculated plants. Variations in terpene profiles were also induced by stem base inoculations and ozone treatment. Ozone might negatively affect terpene defences making plants more susceptible to pathogens and insects.

Effect of benfluralin on Pinus pinea seedlings mycorrhized with Pisolithus tinctorius and Suillus bellinii – Study of plant antioxidant response

In this study, Pinus pinea seedlings mycorrhized with selected ectomycorrhizal fungi (ECMF), Pisolithus tinctorius and Suillus bellinii, were exposed to the herbicide benfluralin. Non-mycorrhized P. pinea seedlings and seedlings mycorrhized with ECMF were transferred to benfluralin-spiked soils at levels of 0.165, 1.65 and 16.5mgkg−1. Plant growth and the fungal role on plant antioxidant response were assessed. In the presence of benfluralin, higher plant growth was observed in mycorrhized plants compared to non-mycorrhized plants, but ECMF colonisation and nutrient uptake were affected by the herbicide. Benfluralin showed no effect on lipid peroxidation in P. pinea seedlings. However, seedlings mycorrhized with S. bellinii showed higher levels of lipid peroxidation when compared to non-mycorrhized ones, both in the presence and absence of benfluralin. The increase of lipid peroxidation could be related to seedling growth induced by the fungus and not to benfluralin toxicity. A similar trend was observed in seedlings mycorrhized with P. tinctorius when exposed to higher benfluralin concentrations, suggesting that the antioxidant response to benfluralin is related not only to fungus species, but also to the level of stress applied in the soil. The higher amount of superoxide dismutase activity in P. pinea seedlings tissues exposed to benfluralin could indicate a plant adaptative response to benfluralin toxicity. Catalase activity showed no increase with benfluralin exposure. Pre-established P. tinctorius mycorrhization conferred root protection and enhanced plant growth in benfluralin spiked soil, inferring that P. tinctorius – P. pinea association could advantageous for plant growth in soils contaminated with pesticides.

Inoculation of Pinus pinea seedlings with Pisolithus tinctorius and Suillus bellinii promotes plant growth in benfluralin contaminated soil

In order to develop new strategies aiming at reconverting agriculture-managed soils into forest plantations, pesticides effect on plant growth needs to be further understood. Thus, the development of Pinus pinea seedlings on soil spiked with benfluralin mimicking different application rates was evaluated, as a well as its effect on root colonisation with Pisolithus tinctorius and Suillus bellinii. P. pinea development and ectomycorrhiza colonization on soil spiked with benfluralin were assessed after 6 month growth. P. pinea seedlings inoculated with P. tinctorius showed higher plant growth and increased nutrient levels than seedlings inoculated with S. bellinii. At field application rate, benfluralin had an inhibitory effect on growth of non inoculated plants. Inoculation with P. tinctorius promoted plant development and nutrient uptake whereas inoculation with S. bellinii did not seem to confer plant protection against the toxic. Although P. tinctorius and S. bellinii were able to form ectomycorrhizae in the presence of benfluralin, the extent of root colonisation was affected by the herbicide. P. tinctorius conferred protection to P. pinea against benfluralin toxicity, overcoming the effect of the herbicide on P. pinea growth and nutrient uptake. This approach can be advantageous for plant establishment on pesticide contaminated soils.

Nitrogen nutrition and drought hardening exert opposite effects on the stress tolerance of Pinus pinea L. seedlings

Functional attributes determine the survival and growth of planted seedlings in reforestation projects. Nitrogen (N) and water are important resources in the cultivation of forest species, which have a strong effect on plant functional traits. We analyzed the influence of N nutrition on drought acclimation of Pinus pinea L. seedlings. Specifically, we addressed if high N fertilization reduces drought and frost tolerance of seedlings and whether drought hardening reverses the effect of high N fertilization on stress tolerance. Seedlings were grown under two N fertilization regimes (6 and 100 mg N per plant) and subjected to three drought-hardening levels (well-watered, moderate and strong hardening). Water relations, gas exchange, frost damage, N concentration and growth at the end of the drought-hardening period, and survival and growth of seedlings under controlled xeric and mesic outplanting conditions were measured. Relative to low-N plants, high-N plants were larger, had higher stomatal conductance (27%), residual transpiration (11%) and new root growth capacity and closed stomata at higher water potential. However, high N fertilization also increased frost damage (24%) and decreased plasmalemma stability to dehydration (9%). Drought hardening reversed to a great extent the reduction in stress tolerance caused by high N fertilization as it decreased frost damage, stomatal conductance and residual transpiration by 21, 31 and 24%, respectively, and increased plasmalemma stability to dehydration (8%). Drought hardening increased tissue non-structural carbohydrates and N concentration, especially in high-fertilized plants. Frost damage was positively related to the stability of plasmalemma to dehydration (r = 0.92) and both traits were negatively related to the concentration of reducing soluble sugars. No differences existed between moderate and strong drought-hardening treatments. Neither N nutrition nor drought hardening had any clear effect on seedling performance under xeric outplanting conditions. However, fertilization increased growth under mesic conditions, whereas drought hardening decreased growth. We conclude that drought hardening and N fertilization applied under typical container nursery operational conditions exert opposite effects on the physiological stress tolerance of P. pinea seedlings. While drought hardening increases overall stress tolerance, N nutrition reduces it and yet has no effect on the drought acclimation capacity of seedlings.

Behaviour of Heterobasidion annosum and Heterobasidion irregulare isolates from central Italy in inoculated Pinus pinea seedlings

Five Heterobasidion annosum and five H. irregulare isolates, collected in central Italy, were tested in order to assess the differences in their behaviour, on PDA medium and in two inoculation experiments on three-year-old Pinus pinea seedlings. In vitro the average growth rate of H. irregulare isolates was faster than H. annosum isolates at a temperature ranging between 5 to 30°C. In an inoculation test, performed at a temperature between 8 and 12°C, all the isolates had a comparable growth in the seedlings after three weeks, but successful infection was significantly more frequent with the H. irregulare isolates. Inoculation was repeated with only two individual isolates, collected from within the National Park of Circeo in disease centres that were very close to each other. This test was performed at a higher temperature (8–24°C), and observations were carried out one, two, three and six weeks later. The H. irregulare isolate always produced a more extended infection in seedlings than the H. annosum isolate. The study indicates that H. irregulare has a somewhat different behaviour versus P. pinea in comparison with H. annosum. We thus believe that the need to monitor how this situation evolves should not be overlooked.

Influence of the ectomycorrhizal fungus Laccaria laccata on pre-emergence, post-emergence and late damping-off by Fusarium oxysporum and F. verticillioides on Stone pine seedlings

In greenhouse experiments, the ectomycorrhizal fungus Laccaria laccata was evaluated for biological control of preemergence, post-emergence and late damping-off of Pinus pinea caused by Fusarium verticillioides and F. oxysporum. In pre-emergence damping-off assays, preinoculation with Laccaria laccata did not significantly improve germination of seeds and no statistical significant differences were found in Fusarium treatments when compared with controls. At 18 weeks after sowing, inoculation with L. laccata reduced the incidence of post-emergence damping-off but differences were significant only in F. oxysporum treatments. Pinus pinea transplanted plants were used in late damping off assays, and only F. oxysporum produced significant damage. Inoculation with L. laccata did not attenuate significantly the virulence of F. oxysporum. However, the percentage of mycorrhization did not reached significant level, so the amount of mycorrhizal fungus was insufficient for effective protection. Although very low percentages of mycorrhization were recorded in all mycorrhized treatments, and Fusarium occurrence significantly reduced mycorrhization, those levels have been efficient to reduce damage in F. oxysporum post-emergence damping-off assays. In short, pre-emergence damping-off was not found; only F. oxysporum produced significant damage on P. pinea seedlings and L. laccata reduced damage when the percentage of mycorrhization reached a significant level. These results have been compared with previous work on P. sylvestris inoculated with the same mycorrhizae isolate and Fusarium pathogens.

Fire reduces Pinus pinea distribution in the northeastern Iberian Peninsula

This study analyzes the recovery of Pinus pinea in forested areas that have burned in the last 30 years. The main objectives of the study were (i) to analyze the success of the regeneration of P. pinea in areas with the presence or absence of surviving adult trees and (ii) to investigate whether or not the post-fire response of P. pinea may affect its present distribution. We analyzed the survival of adult P. pinea trees and the regeneration of P. pinea seedlings in 8 areas burned by wildfires in Catalonia (northeast Spain) between 1979 and 2001. This study shows that the inability of seedlings to establish after fire, together with the short seed dispersal distance of this species and the low level of seedling survival, renders the natural regeneration of P. pinea in burned areas difficult. A comparison of the presence of P. pinea in plots located in burned and unburned areas throughout its distribution area in Catalonia indicates that its presence at the regional level is clearly reduced after fire. Moreover, in the areas where it persists, tree density and cover are smaller than in unburned areas. This decrease of P. pinea forests is exacerbated by the increase in surface area burned by forest fires during recent decades in the Mediterranean basin and by decreasing profits associated with this species as a consequence of the falling value of its pine kernel.

Quantitative detection of Lactarius deliciosus extraradical soil mycelium by real-time PCR and its application in the study of fungal persistence and interspecific competition

Real-Time PCR has been applied to quantify extraradical soil mycelium of the edible ectomycorrhizal fungus Lactarius deliciosus in an interspecific competition experiment under greenhouse conditions. Couples of Pinus pinea seedlings inoculated with either L. deliciosus, Rhizopogon roseolus, or non-inoculated (control) were transplanted into pots filled with two types of soil in all the possible combinations. Total DNA was extracted from soil samples at 3 and 6 months after transplantation to perform real-time PCR analysis. DNA extractions from soil mixed with known amounts of mycelium of L. deliciosus were used as standards. Six months after transplantation, the percentage of mycorrhizas of L. deliciosus and seedling growth were significantly affected by the soil type. Extraradical soil mycelium of L. deliciosus was positively correlated with the final percentage of mycorrhizas and significantly affected by the sampling time and soil depth. The competition effect of R. roseolus was not significant for any of the measured parameters, probably due to the sharp decrease of the mycorrhizal colonization by this fungus. We conclude that real-time PCR is a powerful technique for extraradical mycelium quantification in studies aimed at evaluating the persistence of introduced strains of L. deliciosus in field plantations.

The use of compost in afforestation of Mediterranean areas: Effects on soil properties and young tree seedlings

In Mediterranean frequently burnt areas, fire and erosion result in the decrease of soil fertility, so afforestation is a major concern. We carried out an in situ experiment of compost amendment to improve survival and growth of planted tree seedlings. One-year-tree seedlings of native species (Quercus ilex, Pinus halepensis and Pinus pinea) were planted on a frequently burnt calcareous site. Three rates of fresh co-composted sewage sludge and greenwastes (control without compost, 20 and 40 kg m−2 of compost) were incorporated into the soil at each seedling stem. Changes of soil properties and tree development were studied during 3 years (2001–2003) and 2 years (2002–2003) respectively.The compost improved survival of Quercus ilex and Pinus pinea seedlings in severe drought conditions, but had no effect on Pinus halepensis. For all species seedling length and radial growth and NPK nutrition were increased for both rates of amendment. Amendment improved soil fertility, but available P concentration increased 13 fold in the neighbouring soil of seedlings amended at the maximal rate compared to control. However, amendment did not significantly increase concentrations of Cd, Cr, Ni and Pb in soils or tree seedlings. It increased Cu and Zn total and available concentrations in soils, while foliar Cu and Zn concentrations in the seedlings remained similar in all plots.Compost can efficiently help afforestation of dry soils with low organic matter content. However, sewage sludge concentrations in P, and to a lesser extent in Cu and Zn, limit rates of application that can be applied without environmental hazard.

Container characteristics influence Pinus pinea seedling development in the nursery and field

The growth and development of Pinus pinea seedlings grown in different containers was followed through one growing season in the nursery and 3 years following outplanting in the field. The variables studied in the nursery were height, diameter, biomass of shoots and roots, nutrient uptake and root density. The measured field variables, height and diameter increment and survival, were correlated with the nursery variables. Container volume had the greatest influence on plant morphology. Containers with larger rooting volume had seedlings with larger height and diameter, greater nutrient content, and better field performance. Growing density was correlated with seedling morphology and nutrient concentration in the nursery. Among the variables that influenced container volume, the diameter of the container was the most important, while the depth of the container had a minor influence on seedling morphology. The best indicator of seedling development in the nursery was the ratio of container depth to container diameter, and the optimum ratio was 4. All containers produced seedlings with some root spiralling, including those containers with ribs. There was no relationship between either the number of spiralling roots or the angle of spiralling and container characteristics. Furthermore, root spiralling did not influence seedling performance following outplanting. Root density (root biomass/cm 3) was inversely correlated with container volume but there was no correlation with either depth or growing density. The largest plants were produced with container volumes of 300–400 cm 3, depth/diameter ratios of 4, and growing densities of 200–300 seedlings/m 2. These growing conditions will result in larger Pinus pinea seedlings coming out of the nursery, which will increase growth following outplanting.

Dominance of the mycorrhizal fungus Rhizopogon rubescens in a plantation of Pinus pinea seedlings inoculated with Suillus collinitus

We examined the below-ground mycorrhizal diversity of P. pinea seedlings inoculated with S. collinitus six years after out- planting in a disturbed site, located at La Petite-Camargue (Gard, France). This was performed using the polymerase chain reaction (PCR), the restriction fragment length polymorphism (RFLP) and the sequencing of the nuclear ribosomal internal transcribed spacer (ITS). Seven and one plants corresponding, respectively, to inoculated and non-inoculated (control) treatments were chosen randomly. Examinations were carried out directly from ectomycorrhizae. A total number of 233 root tips was examined. Five ITS RFLP taxa were detected. The ITS taxon I corresponded to the ectomycorrhizal species Rhizopogon rubescens. This fungus was abundant (55%) in the P. pinea ectomycorrhizal community. The other ITS taxa were rare and remained unidentified. The current P. pinea plantation showed a restricted diversity of the ectomycorrhizal community which is typical of ectomycorrhizal communities in young plantations established in disturbed stands.