Single-Leaf Resolution of the Temporal Population Dynamics of Aureobasidium pullulans on Apple Leaves

The abundance of phylloplane microorganisms typically varies over several orders of magnitude among leaves sampled concurrently. Because the methods traditionally used to sample leaves are destructive, it has remained unclear whether this high variability is due to fixed differences in habitat quality among leaves or to asynchronous temporal variation in the microbial population density on individual leaves. We developed a novel semidestructive assay to repeatedly sample the same apple leaves from orchard trees over time by removing progressively more proximal ~1-cm-wide transverse segments. Aureobasidium pullulans densities were determined by standard leaf homogenization and plating procedures and were expressed as CFU per square centimeter of segment. The A. pullulans population densities among leaves were lognormally distributed. The variability in A. pullulans population densities among subsections of a given leaf was one-third to one-ninth the variability among whole leaves harvested concurrently. Sequential harvesting of leaf segments did not result in detectable changes in A. pullulans density on residual leaf surfaces. These findings implied that we could infer whole-leaf A. pullulans densities over time by using partial leaves. When this successive sampling regimen was applied over the course of multiple 7- to 8-day experiments, the among-leaf effects were virtually always the predominant source of variance in A. pullulans density estimates. Changes in A. pullulans density tended to be synchronous among leaves, such that the rank order of leaves arrayed with respect to A. pullulans density was largely maintained through time. Occasional periods of asynchrony were observed, but idiosyncratic changes in A. pullulans density did not contribute appreciably to variation in the distribution of populations among leaves. This suggests that persistent differences in habitat (leaf) quality are primarily responsible for the variation in A. pullulans density among leaves in nature.     

Mould susceptibility of Scots pine (Pinus sylvestris L.) sapwood: Impact of drying,thermal modification,and copper-based preservative

The development of mould on wood surfaces depends on several factors. Although mould does not affect the mechanical properties of wood, it greatly reduces the aesthetic value of wood such as the sapwood of Scots pine (Pinus sylvestris L.), which is very prone to mould. In addition, adverse health effects of mould on humans are a great concern. Different types of dried and treated wood were used to observe whether they had enhanced durability against mould following an accelerated laboratory test method in a climate chamber. Samples were green, air-dried, industrially thermally modified, treated with copper-based preservative, and kiln-dried wood, which were tested within a single test run. The test produced the following main results: The thermal modification increased the durability of the wood, and the protective effectiveness of alternative treatments was comparable to that of commercially available copper-based treatment. However, the initial moisture content of the samples during mould exposure had a great influence on the onset of mould growth. The risk of mould susceptibility of industrial kiln-dried lumber can be reduced by drying using the double-layering technique and planing off the nutrient enriched evaporation surfaces.     

Microtiter Plate Assay for Assessment of Listeria monocytogenes Biofilm Formation

Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32°C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.     

Steam explosion lignins; their extraction,structure and potential as feedstock for biodiesel and chemicals

In the present study, a steam explosion wood pre-treatment process, optimized earlier with respect to ethanol production, has been applied to both softwoods (Picea abies and Pinus sylvestris) and hardwoods (Betula verrucosa and Populus tremula). The alkaline extractable lignins have then been isolated to investigate lignin separation efficiency and lignin structure and to evaluate their potential for producing value-added products, such as biodiesel components or chemicals, in terms of the purity, molecular size, functional groups, β-O-4′ inter-unit linkage content, and degradability in a subsequent processing treatment. The mechanism of lignin modification and possible improvements to the steam explosion pre-treatment process are discussed.     

Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.     

The growth and nutrients status of conifers on ash-treated cutaway peatland

Key message

Use of wood ash or a mixture of wood and oil shale ashes increases the concentrations of P and K in the assimilation organs of conifers and stimulates tree growth.

Abstract

The effect of fertilization with wood ash (10 and 15 t ha^?1) and a mixture of wood ash (10 t ha^?1) and oil shale ash (8 t ha^?1) on the growth (height, root collar diameter, biomass, biomass production) and nutrient concentrations in subsoil and needles of young Pinus sylvestris and Picea abies plants on the Puhatu (Northeast Estonia) cutaway peatland in the first 2 years were studied. After the second growing year differences in the average height growth of P. abies and P. sylvestris were statistically significantly higher on ash-treated plots than on the control plots (p < 0.05), being respectively 1.4–1.6 and 1.5–1.7 times greater than height growth of the control trees. The best results on root collar diameter were observed on mixture ash treatments: the root collars were 1.9 (P. abies) and 2.2 (P. sylvestris) times larger than of the control trees. The biomass of the two conifer species and the biomass production of P. sylvestris in 2012 was the greatest on the mixture ash treatments. Five months after fertilization with ashes the concentrations of P, K, Ca and Mg were higher on the treated plots than on the control plot. Although the concentrations of P and K in P. sylvestris needles rose after the treatment with ash, seedlings suffered from P and K deficiency. The concentrations of P and K in P. abies needles were on optimum. The P/N and the K/N ratios in needles were also improved compared to control trees needles.     

Volatile Emissions from an Epiphytic Fungus are Semiochemicals for Eusocial Wasps

Microbes are ubiquitous on plant surfaces. However, interactions between epiphytic microbes and arthropods are rarely considered as a factor that affects arthropod behaviors. Here, volatile emissions from an epiphytic fungus were investigated as semiochemical attractants for two eusocial wasps. The fungus Aureobasidium pullulans was isolated from apples, and the volatile compounds emitted by fungal colonies were quantified. The attractiveness of fungal colonies and fungal volatiles to social wasps (Vespula spp.) were experimentally tested in the field. Three important findings emerged: (1) traps baited with A. pullulans caught 2750?% more wasps on average than unbaited control traps; (2) the major headspace volatiles emitted by A. pullulans were 2-methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethyl alcohol; and (3) a synthetic blend of fungal volatiles attracted 4,933?% more wasps on average than unbaited controls. Wasps were most attracted to 2-methyl-1-butanol. The primary wasp species attracted to fungal volatiles were the western yellowjacket (Vespula pensylvanica) and the German yellowjacket (V. germanica), and both species externally vectored A. pullulans. This is the first study to link microbial volatile emissions with eusocial wasp behaviors, and these experiments indicate that volatile compounds emitted by an epiphytic fungus can be responsible for wasp attraction. This work implicates epiphytic microbes as important components in the community ecology of some eusocial hymenopterans, and fungal emissions may signal suitable nutrient sources to foraging wasps. Our experiments are suggestive of a potential symbiosis, but additional studies are needed to determine if eusocial wasp–fungal associations are widespread, and whether these associations are incidental, facultative, or obligate.     

Defense mechanisms of conifers : relationship of monoterpene cyclase activity to anatomical specialization and oleoresin monoterpene content

Cell-free extracts from Pinus ponderosa Lawson (ponderosa pine) and Pinus sylvestris L. (Scotch pine) wood exhibited high levels of monoterpene synthase (cyclase) activity, whereas bark extracts of these species contained no detectable activity, and they inhibited cyclase activity when added to extracts from wood, unless polyvinylpyrrolidone was included in the preparation. The molecular mass of the polyvinylpyrrolidone added was of little consequence; however, polyvinylpolypyrrolidone (a cross-linked insoluble form of the polymer) was ineffective in protecting enzyme activity. Based on these observations, methods were developed for the efficient extraction and assay of monoterpene cyclase activity from conifer stem (wood and bark) tissue. The level of monoterpene cyclase activity for a given conifer species was shown to correlate closely with the monoterpene content of the oleoresin and with the degree of anatomical complexity of the specialized resin-secreting structures. Cyclase activity and monoterpene content were lowest in the stems of species containing only isolated resin cells, such as western red cedar (Thuja plicata D. Don). Increasing levels of cyclase activity and oleoresin monoterpenes were observed in advancing from species with multicellular resin blisters (true firs [Abies]) to those with organized resin passages, such as western larch (Larix occidentalis Nutt.), Colorado blue spruce (Picea pungens Engelm.) and Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The highest levels of cyclase activity and oleoresin monoterpenes were noted in Pinus species that contain the most highly developed resin duct systems. The relationship between biosynthetic capacity, as measured by cyclase activity, monoterpene content, and the degree of organization of the secretory structures for a given species, may reflect the total number of specialized resin-producing cells per unit mass of stem tissue.     

Adaptive Responses of Morphological Forms of the Pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.) under Stressful Conditions of the Northern Taiga (in the Northern Dvina Basin)

The variability of physiological and biochemical indicators and radial growth of different forms of Pinus sylvestris L. f. (var.) sulfuranthera Kozubow and f. (var.) erythranthera Sanio under flooding conditions has been studied. It is shown that the dynamics of photosynthetic pigments (chlorophylls and carotenoids), proline, proteins, ascorbic acid, peroxidase activity, and the light-harvesting complex in the pine needles depend on meteorologic factors and the phenophase. Forms with different colors of anthers differ in seasonal dynamics of the content of stressful metabolites and age variability of the radial growth of wood. Our results indicate that different forms of Pinus sylvestris L. have nuances in adaptation to stress conditions.     

First description of Candida nivariensis in Brazil: antifungal susceptibility profile and potential virulence attributes

This study evaluated the antifungal susceptibility profile and the production of potential virulence attributes in a clinical strain of Candida nivariensis for the first time in Brazil, as identified by sequencing the internal transcribed spacer (ITS)1-5.8S-ITS2 region and D1/D2 domains of the 28S of the rDNA. For comparative purposes, tests were also performed with reference strains. All strains presented low planktonic minimal inhibitory concentrations (PMICs) to amphotericin B (AMB), caspofungin (CAS), and voriconazole. However, our strain showed elevated planktonic MICs to posaconazole (POS) and itraconazole, in addition to fluconazole resistance. Adherence to inert surfaces was conducted onto glass and polystyrene. The biofilm formation and antifungal susceptibility on biofilm-growing cells were evaluated by crystal violet staining and a XTT reduction assay. All fungal strains were able to bind both tested surfaces and form biofilm, with a binding preference to polystyrene (p < 0.001). AMB promoted significant reductions (≈50%) in biofilm production by our C. nivariensis strain using both methodologies. This reduction was also observed for CAS and POS, but only in the XTT assay. All strains were excellent protease producers and moderate phytase producers, but lipases were not detected. This study reinforces the pathogenic potential of C. nivariensis and its possible resistance profile to the azolic drugs generally used for candidiasis management.     

Direct production of feruloyl oligosaccharides and hemicellulase inducement and distribution in a newly isolated Aureobasidium pullulans strain

Studies were carried out to screen and identify strains that are able to directly produce ferulic oligosaccharides (FOs) from wheat bran (WB). The inducement and distribution of hemicellulases from strain 2012, which was identified as a non-melanin secreting strain of Aureobasidium pullulans (A. pullulans), were also determined. In a 60 g/L WB solution, A. pullulans could produce 545 nmol/L FOs, 64.12 IU/mL xylanase and 0.14 IU/mL ferulic acid esterase (FAE). A. pullulans was cultivated in media with WB, glucose, xylose, sucrose, lactose or xylan as the carbon source, and hemicellulases were mainly induced by xylan and WB and inhibited by glucose and sucrose. Xylanase and FAE were mainly present in the culture filtrate, xylosidase in the hyphal filaments and arabinofuranosidase was a membrane-bound enzyme. The yield of FOs was positively correlated to the hemicellulases activity, and significantly positively (P < 0.05) correlated to the xylanase activity (r = 0.992).     

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).^1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.^3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.^4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.^18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),^8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),^9,10 and Bacteroidetes (Bac303).¹¹ In addition, specific probes binding to Streptococcus mutans (MUT590)^12,13 and Porphyromonas gingivalis (POGI)^13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.^5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations^3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.     

Biofilm development on leaf and wood surfaces in a boreal river

SUMMARY 1. Biofilms are organic layers that develop on submerged surfaces. They are composed of micro-organisms, exoenzymes, and detritus particles enclosed within a gelatinous matrix. While much is known about mineral surface biofilms, those developing on organic surfaces have not been extensively studied. We examined the influences of current velocity and substratum composition on biofilm development in a fourth-order North American boreal river. 2. Arrays of white birch ice-cream sticks and sugar maple leaves were placed at fast and slow current sites. Samples were collected periodically, analysed for mass loss, and assayed for microbial biomass (ATP, ergosterol, chlorophyll a) and exoenzyme activity associated with lignocellulose degradation (exo- and endocellulase, β-glucosidase, phenol oxidase, peroxidase). 3. Biofilms developed rapidly on both surfaces. On leaves, biomass peaked within 30 days of exposure. On wood, ATP and chlorophyll a concentrations peaked within 30–70 days, whereas ergosterol increased throughout the study (161 days). On leaves, current velocity had little influence on biofilm development, although breakdown rates were greater at the fast flow site. On wood, ATP and chlorophyll a concentrations were greater at the fast flow site, whereas ergosterol concentrations and breakdown rates were similar at both sites. Microbial biomass was consistently greater on wood than leaves, Exoenzyme activity developed rapidly on both surfaces; current velocity had little influence on activity. Except for β-glucosidase, activities were greater on wood than leaves. 4. Our results suggest that fungi are an important structuring element of organic surface biofilms and the physical stability of the substratum strongly influences biofilm development. Leaf surfaces are susceptible to softening and fragmentation, truncating biofilm development. In contrast, abrasion of wood surfaces removes senescent material exposing fresh substratum for colonization. Thus, wood surfaces with their greater physical stability, permit the development of more extensive biofilms. Wood surfaces may represent an overlooked but important site of metabolic activity in streams.     

Effect of Batch and Fed-Batch Growth Modes on Biofilm Formation by <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> at Different Temperatures

The influence of Listeria monocytogenes (L. monocytogenes) biofilm formation feeding conditions (batch and fed-batch) at different temperatures on biofilm biomass and activity was determined. Biofilm biomass and cellular metabolic activity were assessed by Crystal Violet (CV) staining and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) colorimetric method, respectively. Live/Dead staining was also performed in order to get microscopic visualization of the different biofilms. Results revealed that at refrigeration temperature (4°C) a higher amount of biofilm was produced when batch conditions were applied, while at higher temperatures the fed-batch feeding condition was the most effective on biofilm formation. Moreover, independently of the temperature used, biofilms formed under fed-batch conditions were metabolically more active than those formed in batch mode. In conclusion, this work shows that different growth modes significantly influence L. monocytogenes biofilm formation on abiotic surfaces as well as the metabolic activity of cells within biofilms.     

Macroinvertebrate colonization and biofilm development on leaves and wood in a boreal river

• 1 Biofilms, the accumulation of micro-organisms, exoenzymes, and detritus particles on submerged surfaces, may change the quality of leaves and wood as sources of food and/or habitat for invertebrates. We examined the relationship between macroinvertebrate assemblages and biofilm development on leaves and wood in a boreal river.
• 2 Arrays of white birch ice-cream sticks and sugar maple leaves were placed at fast- and slow-current sites. Samples were collected periodically, and assayed for microbial biomass (ATP, ergosterol, chlorophyll a) and macroinvertebrate colonizers.
• 3 Microbial biomass and macroinvertebrate density were consistently greater on wood than leaves. Taxon richness was similar for all substratum/current combinations, but taxon density (number of taxa m^?2) was greater on wood. Macroinvertebrate density and taxon richness correlated with all microbial indicators when data from both substrata were pooled.
• 4 Leaves did not support as great a density of invertebrates as wood perhaps because of their faster breakdown rate and truncated biofilm development. Greater stability and the potential for surface complexity may make wood a site of higher macroinvertebrate diversity than leaves.

     

Shewanella putrefaciens Adhesion and Biofilm Formation on Food Processing Surfaces

Laboratory model systems were developed for studying Shewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25°C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 10⁸ CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 10² CFU/cm²) than in a batch system (reaching 10⁷ CFU/cm²), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4′,6′-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.     

Growth and characterization of Escherichia coli DH5α biofilm on concrete surfaces as a protective layer against microbiologically influenced concrete deterioration (MICD)

Biofilms of selected bacteria strains were previously used on metal coupons as a protective layer against microbiologically influenced corrosion of metals. Unlike metal surfaces, concrete surfaces present a hostile environment for growing a protective biofilm. The main objective of this research was to investigate whether a beneficial biofilm can be successfully grown on mortar surfaces. Escherichia coli DH5α biofilm was grown on mortar surfaces for 8 days, and the structure and characteristics of the biofilm were studied using advanced microscopy techniques such as scanning electron microscopy and confocal laser scanning microscopy in combination with fluorescence in situ hybridization, live/dead, extracellular polymer staining, ATP analysis, and membrane filtration. A biofilm layer with a varying thickness of 20–40 μm was observed on the mortar surface. The distribution of live and dead bacteria and extracellular polymers varied with depth. The density of the live population near the mortar surface was the lowest. The bacteria reached their highest density at three fourths of the biofilm depth and then decreased again near the biofilm–liquid interface. Overall, the results indicated a healthy biofilm growth in the chosen growth period of 8 days, and it is expected that longer growth periods would lead to formation of a more resistant biofilm with more coverage of mortar surfaces.     

Influence of microbial interactions and EPS/polysaccharide composition on nutrient removal activity in biofilms formed by strains found in wastewater treatment systems

The study of biofilm function, structure and microbial interactions might help to improve our understanding of biofilm wastewater treatment processes. However, few reports specifically address the influence of interactions within multispecies biofilms on microbial activity and biofilm composition. Thus, the relationship between biofilm formation, denitrification activity, phosphorus removal and the composition of extracellular polymeric substances (EPS), exopolysaccharides and the bacterial community was investigated using biofilms of denitrifying and phosphorus removing strains Comamonas denitrificans 110, Brachymonas denitrificans B79, Aeromonas hydrophila L6 and Acinetobacter calcoaceticus ATCC23055. Denitrification activity within the biofilms generally increased with the amount of biofilm while phosphorus removal depended on bacterial growth rate. Synergistic effects of co-growth on denitrification (B. denitrificans B79 and A. hydrophila L6) and phosphorus removal (C. denitrificans 110 with either A. calcoaceticus or A. hydrophila L6) were observed. B. denitrificans B79 was highly affected by interspecies interactions with respect to biofilm formation, denitrification activity and EPS composition, while C. denitrificans 110 remained largely unaffected. In some of the dual and quadruple strain biofilms new exopolysaccharide monomers were detected which were not present in the pure strain samples.     

Comparison based on field tests of three low-environmental-impact wood treatments

In order to promote the use of Pinus pinea L. wood within the Migliarino-San Rossore Nature Reserve (Pisa, central Italy), three low-environmental-impact wood treatments, supposed to enhance natural durability, were compared. Impregnations with an oil-based preservative and natural waxes and a wood thermal treatment were tested in the field in accordance with standards ENV 12037 and EN 252. The above-ground test revealed that: P. pinea sapwood is more durable than Pinus sylvestris sapwood; all the alternative treatments showed a low mean decay level; wax and oil treatments performed as well as the traditional copper-based preservative; the natural durability class of P. pinea will only be calculated upon the complete failure of all reference lap-joints. The main outcomes from the in-ground test were: All the tested treatments increased the durability of wood, and the protective effectiveness of alternative treatments was comparable to traditional copper-based ones, or even superior in the case of heated oil. Taking certain mechanical and aesthetic limitations into account, all the treatments were suitable for the promotion of the P. pinea wood commodity in use classes 3 and 4.