Heartwood extractives from the Amazonian trees <Emphasis Type="Italic">Dipteryx odorata,Hymenaea courbaril</Emphasis>, and <Emphasis Type="Italic">Astronium lecointei</Emphasis> and their antioxidant activities

Heartwood extracts from Amazonian trees cumaru-ferro (Dipteryx odorata), jatoba (Hymenaea courbaril), and guarita (Astronium lecointei) exhibit antioxidant activities comparable with that of α-tocopherol, a well-known antioxidant. This article reports the characterization of the antioxidant compounds in the extracts of the three heartwoods. Silica gel column chromatography of the cumaru-ferro EtOAc extract yielded (−)-(3R)-7,2′,3′-trihydroxy-4′-methoxyisoflavan and (+)-(3R)-8,2′,3′-trihydroxy-7,4′-dimethoxyisoflavan. Silica gel column chromatography followed by preparative high-performance liquid chromatography of the jatoba EtOAc extract yielded (−)-fisetinidol and (+)-trans-taxifolin. Chemical structures were assigned using electron-ionization mass spectrometry, ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy including nuclear Overhauser effect spectroscopy (NOESY), as well as optical rotation and circular dichroism. Gas chromatography-mass spectrometry demonstrated that the isolated compounds were predominant in the EtOAc extracts. In the guarita EtOAc extract, catechin and gallic acid were identified by comparing their retention times and mass fragmentation patterns with those of authentic samples. Antioxidant activity determined by the 1,1-diphenyl-2-picrylhydrazyl assay demonstrated that all these compounds had activities comparable with that of α-tocopherol. Part of this report was presented at the 57th Annual Meeting of the Japan Wood Research Society, Hiroshima, August 2007     

<Emphasis Type="Italic">Ophiostoma</Emphasis> species associated with bark beetles infesting three <Emphasis Type="Italic">Abies</Emphasis> species in Nikko,Japan

Ophiostoma species were isolated from bark beetles and Abies mariesii, A. veitchii and A. homolepis attacked by the beetles in Nikko, Tochigi, central Honshu, Japan. One to two Ophiostoma species were frequently isolated from each species of bark beetle. Ophiostoma subalpinum was the most common associate of Cryphalus montanus. Ophiostoma sp. B as well as O. subalpinum was a common fungus associated with Polygraphus proximus. Ophiostoma europhioides was isolated from Dryocoetes hectographus and D. autographus as one of the common associates. Ophiostoma sp. J and Ophiostoma sp. S were frequently isolated from D. autographus and D. striatus, respectively. These fungi seem to have specific relationships with particular bark beetles. Ophiostoma sp. B, Ophiostoma sp. J and Ophiostoma sp. S have unique morphological characteristics and appear to be new species. Five trees of A. veitchii, approximately 43 years old, were inoculated with five Ophiostoma species to assess the relative virulence of the fungi. Ophiostoma subalpinum, Ophiostoma sp. B, and O. europhioides had relatively higher virulence than the other species studied.     

Aphidicidal activity of some indigenous plant extracts against bean aphid <Emphasis Type="Italic">Aphis </Emphasis><Emphasis Type="Italic">craccivora</Emphasis> Koch (Homoptera: Aphididae)

Aphidicidal activity of hot and cold water extracts of some indigenous plants, Azadirachta indica A. Juss (neem), Calotropis procera (Aiton) W.T. Aiton (akanda), Polygonum hydropiper L. (biskatali) and Ipomoea sepiaria J. Koenig ex Roxb. (bankalmi), were tested against the bean aphid, Aphis craccivora Koch. Hot water extract of P. hydropiper and A. indica was found to be the most effective (87.6–94.5 and 80.47–89.6% mortality respectively, P < 0.01) among all the extracts. Other hot and cold water extracts also appeared to be useful (59.5–87.5% mortality) as pesticides for A. craccivora. The highest yield (3.25 kg per plant) was obtained using hot water extract of P. hydropiper followed by hot water extract of A. indica (3.15 kg per plant). The lowest yield (0.32 kg per plant) was recorded from the control block. All the phytoproduct treatments had significantly (P < 0.01) better yield than the control block.     

Antioxidant compounds from <Emphasis Type="Italic">Eucalyptus grandis</Emphasis> biomass by subcritical liquid water extraction

The effectiveness of subcritical liquid water processing for extraction of antioxidants from the leaves of Eucalyptus grandis biomass was evaluated by determining the capability of the processed samples to scavenge peroxynitrite radicals in vitro, as compared with the extracts obtained by conventional extraction. Pyrogallol (1), 5-hydroxymethyl-2-furaldehyde (2), and 4,4,6,6-tetramethyl-3,5-dioxo-cyclohex-1-enecarboxylic acid (3) were identified as the major products obtained from the subcritical liquid water extracts. Among these, pyrogallol (1) exhibited stronger antioxidant activity than butylated hydroxytoluene, which was used as a standard for the antioxidant activity measurement. This research was presented at the conference of the Japan Society for Bioscience, Biotechnology, and Agrochemistry (JSBBA) in Tokyo, March 2007.     

Biological performance of <Emphasis Type="Italic">Eucalyptus camaldulensis</Emphasis> leaf oils from Thailand against the subterranean termite <Emphasis Type="Italic">Coptotermes formosanus</Emphasis> Shiraki

The antitermitic activities of leaf oils and their constituents, taken from three clones of Eucalyptus camaldulensis Dehnh. in Thailand, against Coptotermes formosanus Shiraki were investigated in contact and noncontact tests. The termiticidal mechanism was also examined. Antitermitic tests demonstrated that E. camaldulensis leaf oils were both contact toxicants and fumigants to C. formosanus with LC₅₀ values ranging between 12.68 and 17.50 mg/g by the contact method, and between 12.65 and 17.50 mg/petri dish (100 cm³) by the noncontact method. p-Cymene and γ-terpinene were primarily responsible for the contact toxicity and 1,8-cineole was responsible for fumigation. From the investigation of termiticidal mechanism, E. camaldulensis leaf oils exhibited the inhibition of acetylcholinesterase activity and showed the common symptoms of a neurotoxic mode of action against C. formosanus. Part of this report was presented at the 58th Annual Meeting of the Japan Wood Research Society, Tsukuba, March 2008     

<Emphasis Type="Italic">Bursaphelenchus mucronatus</Emphasis> (Nematoda: Aphelenchoididae) vectored by <Emphasis Type="Italic">Monochamus </Emphasis><Emphasis Type="Italic">urussovi</Emphasis> (Coleoptera: Cerambycidae) in Hokkaido,Japan

Bursaphelenchus mucronatus Mamiya et Enda has been recovered for the first time from adults of the cerambycid beetle, Monochamus urussovi (Fischer), in Hokkaido, Japan. The nematode was also recovered from the inner bark of Picea jezoensis (Siebold et Zuccarini) Carrière and Abies sachalinensis (Fr Schmidt) Masters infested with M. urussovi larvae. PCR–RFLP analysis indicated that B. mucronatus in Hokkaido is the European type.     

Bioremediation of CCA-treated wood by brown-rot fungi <Emphasis Type="Italic">Fomitopsis palustris</Emphasis>, <Emphasis Type="Italic">Coniophora puteana</Emphasis>, and <Emphasis Type="Italic">Laetiporus sulphureus</Emphasis>

This study evaluated oxalic acid accumulation and bioremediation of chromated copper arsenate (CCA)-treated wood by three brown-rot fungi Fomitopsis palustris, Coniophora puteana, and Laetiporus sulphureus. The fungi were first cultivated in a fermentation broth to accumulate oxalic acid. Bioremediation of CCA-treated wood was then carried out by leaching of heavy metals with oxalic acid over a 10-day fermentation period. Higher amounts of oxalic acid were produced by F. palustris and L. sulphureus compared with C. puteana. After 10-day fermentation, oxalic acid accumulation reached 4.2 g/l and 3.2 g/l for these fungi, respectively. Fomitopsis palustris and L. sulphureus exposed to CCA-treated sawdust for 10 days showed a decrease in arsenic of 100% and 85%, respectively; however, C. puteana remediation removed only 18% arsenic from CCA-treated sawdust. Likewise, chromium removal in F. palustris and L. sulphureus remediation processes was higher than those for C. puteana. This was attributed to low oxalic acid accumulation. These results suggest that F. palustris and L. sulphureus remediation processes can remove inorganic metal compounds via oxalic acid production by increasing the acidity of the substrate and increasing the solubility of the metals.An erratum to this article can be found at     

Successful <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Populus tomentosa</Emphasis> with apple <Emphasis Type="Italic">SPDS</Emphasis> gene

The problem of salinized soils has become one of the most serious constraints to agricultural and forest productivity. With the purpose of enhancing salt stress tolerance of Populus tomentosa, we transformed this tree species with spermidine synthase （SPDS） genes derived from an apple by an Agrobacterium-mediated method. Four transgenic clones were confu＇med by PCR and Southern blot analysis. As well, the expression of introduced SPDS genes was analyzed by real-time quantitative PCR.     

Characteristic Raman bands for <Emphasis Type="Italic">Artocarpus heterophyllus</Emphasis> heartwood

The Raman spectrum of Artocarpus heterophyllus heartwood, which proved to be a rich source of flavonoids, exhibited two characteristic bands, at 1247cm^–1 and 745cm^–1. The bands also appeared in the Raman spectrum of the yellow-brown needles extracted from the heartwood with methanol. Based on the Raman measurements of flavones and related compounds, it was predicted that the Raman band at 1247cm^–1 may be attributed to flavonoid-type compounds. No vibrational band corresponding to the characteristic Raman bands was observed by diffuse reflectance infrared spectroscopy. Thus, it was suggested that observation of the characteristic bands is an advantage of Fourier transform-Raman spectroscopy for nondestructive analysis of wood.     

Antimicrobial activity against <Emphasis Type="Italic">Streptococcus sobrinus</Emphasis> and glucosyltransferase inhibitory activity of taxifolin and some flavanonol rhamnosides from kempas (<Emphasis Type="Italic">Koompassia malaccensis</Emphasis>) extracts

Twenty plant materials collected from the islands of Java and Kalimantan in Indonesia were extracted with 50% aqueous ethanol (crude extract). The crude extracts were assayed for antimicrobial activities against Streptococcus sobrinus and for glucosyltransferase (GTase) inhibition. Fourteen extracts inhibited the growth of S. sobrinus by more than 50% and six extracts inhibited GTase activity by more than 50% at a concentration of 100 μg/ml. Koompassia malaccensis (kempas) extracts showed 90% depression of S. sobrinus growth and 80% inhibition of GTase activity at a concentration of 100 μg/ml. Kempas crude extracts were subjected to column chromatography using Sephadex LH-20 and then preparative high-performance liquid chromatography to isolate four compounds A, B, C, and D. These compounds were identified as taxifolin and the flavanonol rhamnoside isomers neoastilbin, astilbin, and isoastilbin, respectively, from ¹H and ¹³C nuclear magnetic resonance (NMR) spectra and other two-dimensional NMR techniques (COSY, HMBC, and HMQC). Each compound depressed the growth of S. sobrinus over a concentration range of 9.3242.7 μg/ml and showed GTase inhibitory activity with IC₅₀ values in the range 27.4–57.3 μg/ml. Taxifolin and flavanonol rhamnoside isomers isolated for the first time from kempas could be potent compounds for preventing dental caries. Part of this report was presented at the 57th Annual Meeting of the Japan Wood Research Society Conference, Hiroshima, 2007     

Recessive inheritance of morphological characteristics of utsukushimatsu,<Emphasis Type="Italic">Pinus densiflora</Emphasis> f.<Emphasis Type="Italic">umbraculifera</Emphasis>

Pinus densiflora f.umbraculifera, commonly known as utsukushimatsu, is a distinctively shaped form of Japanese red pine whose growth is restricted to a forest stand in Shiga Prefecture, Japan. The inheritance mode of morphological characteristics of utsukushimatsu was studied to preserve the genetic resource of this pine. As previously reported, F₁ trees grown from open-pollinated seeds harvested from trees inhabiting the native stand showed two phenotypes: one resembling utsukushimatsu, which produces multiple trunks, and the other resembling normalP. densiflora, which produces one or a few trunks. In the present study, controlled pollination was carried out using F₁ and normalP. densiflora trees. Segregation ratios of the two phenotypes observed in the F₂ population showed that the morphological characteristics of utsukushimatsu are inherited recessively. This suggests that the mutation of one gene or a few closely linked genes controls the morphological characteristics of utsukushimatsu. Since multiple trunk formation of utsukushimatsu might be related to a loss of lateral bud inhibition, it follows that a simple gene mutation breaks apical dominance inP. densiflora.     

Distinction of resin compounds between the healthy bark and the resinous stem canker of <Emphasis Type="Italic">Thujopsis dolabrata</Emphasis> var. <Emphasis Type="Italic">hondae</Emphasis>

Methyl esters of higher fatty acids were detected from the healthy bark of Thujopsis dolabrata var. hondae but not from the bark of the resinous stem canker of T. dolabrata var. hondae. This difference enabled us to distinguish healthy trees from diseased ones. Fourteen diterpenes were also isolated from the n-hexane extracts of the bark-glued resin taken from the resinous stem canker of T. dolabrata var. hondae. Of these diterpenes, abietane diterpenes [abieta-7,13-diene (1), abietinol (2), dehydroabietinol (4)], pimarane diterpene [sandaracopimaric acid (8)], and labdane diterpenes [manool (10), torulosol (11), torulosal (12), cupressic acid (13)] were first isolated from T. dolabrata var. hondae.Part of this paper was presented at the 47th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1999     

Antifeedant activities of secondary metabolites from <Emphasis Type="Italic">Ajuga nipponensis</Emphasis> against adult of striped flea beetles, <Emphasis Type="Italic">Phyllotreta striolata</Emphasis>

Antifeedant activities of the isolated chemical compounds from Ajuga nipponensis, were studied against adult of striped leaf beetles. The methanol, petroleum ether, ethyl acetate and n-butanol extracts at 1.0 mg/ml, were used in this study. All four extracts exhibited more than 65 antifeedant index at 24 h and ethyl acetate extract showed significant activity against striped leaf beetles with 83.12 antifeedant index. Six compounds and one fraction were isolated by chromatography and their structures were identified by NMR, MS and FTIR spectra. At 2.0 mg/ml for 24 h the three compounds 20-hydroxyecdysone, acacetin and apigenin showed considerable activities with antifeedant indexes 59.29, 51.22 and 61.55, respectively. In contrast to this the antifeedant indexes of acacetin and apigenin, were sharply reduced as the time extended and that of 20-hydroxyecdysone remained unchanged. In addition, the synergistic effects of two mixtures of secondary metabolites, were studied and no sharp difference was observed.     

Effects of stilbenes from bark of <Emphasis Type="Italic">Picea glehnii</Emphasis> (Sieb. et Zucc) and their related compounds against feeding behaviour of <Emphasis Type="Italic">Reticulitermes speratus</Emphasis> (Kolbe)

The effects of stilbene glucosides and related compounds on termite feeding behavior were investigated using paper disc methods against the subterranean termite Reticulitermes speratus. The stilbene-rich fraction and isorhapontin (3-methoxy-3,4, 5-trihydroxystilbene-3--d-glucoside) from bark extracts of Picea glehnii showed avoidance by termites in choice tests. In the no-choice tests using compounds purified from the stilbene-rich fraction, the largest feeding deterrent effect was observed for piceid (3,4,5-trihydroxystilbene-3--d-glucoside), followed by isorhapontin, and astringin (3,3,4,5-tetrahydroxystilbene-3--d-glucoside), at the concentrations from 0.63 to 2.5µmol/disc. No change in activity was observed at retentions of more than 5.0µmol/disc. When the activities of isorhapontin and its aglycone derivative (isorhapontigenin: 3-methoxy-3,4,5-trihydroxystilbene) were compared with that of taxifolin (3,3,4,5,7-pentahydroxyflavanone) in the no-choice test, the stilbenes exhibited a larger antifeedant potential. Methylation of isorhapontigenin increased its termiticidal activity.Part of this study was presented at the 32nd Annual Meeting of the International Research Group on Wood Preservation, 2001     

Laboratory and field evaluation of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> for controlling Mulberry whitefly <Emphasis Type="Italic">Pealius mori</Emphasis> Takahashi (Homoptera: Aleyrodidae) in mulberry (<Emphasis Type="Italic">Morus alba</Emphasis> Linn)

In a laboratory screening of 12 isolates of entomopathogenic fungi against nymphs of the mulberry whitefly (Pealius mori Takahashi), Beauveria bassiana (Balsamo-Crivelli) Vuillemin CKB-048 was the most virulent, causing 87 ± 3% mortality at 1 × 10⁶ conidia/ml. Infection was confirmed by growth of the fungus from cadavers and by scanning electron microscopy of treated nymphs. Beauveria bassiana CKB-048 was formulated as a wettable powder (1 × 10⁹ conidia/g) and tested in two mulberry (Morus alba Linn) plantations in central and northeastern Thailand. In both locations, two spray applications of B. bassiana CKB-048 at 3.75 × 10¹² to 6.25 × 10¹² conidia/ha and at 14 day intervals provided good control of whitefly nymphs; control with B. bassiana CKB-048 was comparable to that with the pesticide buprofezin at 250 g of active ingredient/ha. In addition, no mortality of silkworm larvae occurred when the larvae were fed with mulberry leaves sprayed with B. bassiana CKB-048 7, 14, or 21 days earlier.     

Radial variations of wood properties of an endangered species, <Emphasis Type="Italic">Pinus armandii</Emphasis> var. <Emphasis Type="Italic">amamiana</Emphasis>

A dead tree of Pinus armandii Franch. var. amamiana (Koidz.) Hatusima (abbreviated to PAAm) was obtained from a natural habitat on Tanega-shima Island and various properties of its wood were investigated. Grain angle was measured and soft X-ray analysis was undertaken to obtain the density in each annual ring. Unit shrinkage and dynamic properties were measured by shrinkage, bending, and compression tests. Variations of wood properties in the radial direction, relationships of wood properties to density, and annual ring width were examined. Roughly speaking, variations in the radial direction of the grain angle, twist angle by drying, Young’s modulus and strength in static bending, absorbed energy in impact bending, compressive Young’s modulus, compressive strength, and compressive proportional limit corresponded to the variation of annual ring width. As a result, it was determined that if PAAm is afforested artificially for the purposes of lumber production and conservation, the annual rings of logs should not be too widely spaced. Wood properties of PAAm were similar to those of Japanese black pine (Pinus thunbergii Parl.), which is another representative pine on Tanegashima Island. This study was presented in part at the 56th Annual Meeting of the Japan Wood Research Society, Hiroshima, August 2007     

Effect of rhizobial inoculation methods and host plant provenances on nodulation and growth of <Emphasis Type="Italic">Acacia senegal</Emphasis> and <Emphasis Type="Italic">Acacia nilotica</Emphasis>

The purpose of this work was to determine the most efficient methods of inoculation to significantly improve nodulation and growth of Acacia senegal and Acacia nilotica, grown under greenhouse conditions. Our results showed that inoculation using dissolved alginate beads containing rhizobia significantly improved the growth of both acacia species better than the growth of plants in other treatments. The experiment with A. nilotica was conducted in two unsterilised soils from different areas. Plants grown in soil from Bel Air were well-nodulated and showed better growth than plants grown in soil from Sangalkam. However, no difference between these soils was shown between the several methods of inoculation and their effect on the nodulation and growth of plants. An interaction between A. senegal and A. nilotica provenances and the effect of inoculation with rhizobia was also demonstrated. Bel Air provenance of A. senegal, Dahra and RIM provenances of A. nilotica grew best of several provenances tested. These results suggest that (1) it may be possible to improve growth and yield of A. senegal and A. nilotica by careful selection of each symbiotic partner; and (2) nursery-grown seedlings of A. senegal and A. nilotica should be inoculated, just after sowing, with dissolved alginate beads containing a mixture of selected rhizobia. Résumé. Lobject de notre travail était de déterminer la méthodologie dinoculation la plus efficace pour améliorer significativement la nodulation et la croissance dAcacia senegal et dAcacia nilotica poussant dans des conditions de serre. Nos résultats ont montré que linoculation utilisant des billes dalginate dissoutes améliorait significativement la croissance des deux espèces dAcacia par rapport à celle des plantes des autres traitements. Lexpérience avec A. nilotica a été faite dans deux types de sol non stériles. Nous avons observé que les plants poussant dans le sol de Bel Air étaient plus nodulés et mieux développés que ceux poussant dans le sol de Sangalkam. Cependant, aucune différence significative entre les deux sols na été mise en évidence en termes deffet des différentes méthodologies dinoculation sur la nodulation et la croissance des plantes. Il a aussi été démontré lexistence dune interaction entre les provenances dA. senegal et dA. nilotica et leffet de linoculation avec rhizobium. Cest ainsi que les provenances Bel Air dA. senegal, Dahra et RIM dA. nilotica ont le mieux poussé parmi les différentes provenances testées. Lensemble de ces résultats suggèrent: ⁽¹⁾ il devrait être possible daméliorer la croissance et la production dA. senegal et dA. nilotica par une sélection des deux partenaires symbiotiques; ⁽²⁾ Les jeunes plantules dA. senegal et dA. nilotica produites en pépinière devraient être inoculées juste après le semis avec des billes dalginate dissoutes contenant un cocktail de souches sélectionnées.     

Spatial pattern of beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) and oak (<Emphasis Type="Italic">Quercus pubescens</Emphasis> Mill.) seedlings in natural pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.) woodlands

Spatial pattern of recruitment is an important factor influencing population dynamics of plant communities. The spatial pattern is determined by seed dispersal and by the spatial variability of germination and initial survival. In the process of forest expansion following farmland abandonment, mid- and late-successional species are often dispersed in pioneer forests by birds. This could result in an aggregated spatial pattern of seeds that could influence the dynamics of these species in mixed pioneer forests. In the sub-Mediterranean area, mid- and late-successional species such as Quercus pubescens (downy oak) and Fagus sylvatica (European beech) are expected to replace pioneer Pinus species. Using a point sampling method we demonstrated that beech and oak seedlings (height <2 m) have a clumped distribution in the understorey of pine. This could result from an aggregated dispersal by jays (dispersal effect) or from preferential recruitment in particular habitats (habitat effect). To test these hypotheses we proposed a statistical analysis of spatial patterns of regeneration of beech and oak. Ground cover variables (i.e. cover by rock outcrops, herbs, box shrubs, mosses or pine) did not differ significantly around seedlings as compared with random sample plots. Likewise, clumped seedlings had growth similar to isolated seedlings, thus refuting the hypothesis of preferential location in the most favourable microsites. Aggregated dispersal seems to be involved in the process of regeneration. Since beech and oak seedlings have contrasting ecological demands, we discuss the implication of this pattern for the replacement dynamics of pine by these species.     

Effectiveness of Neem (<Emphasis Type="Italic">Azadirachta indica</Emphasis>) insecticides against Brassica pod midge (<Emphasis Type="Italic">Dasineura brassicae</Emphasis> Winn<Emphasis Type="Italic">.</Emphasis>)

The efficiency of the botanical insecticide (BI) NeemAzal T/S (containing 1% Azadirachtin A), on the basis of azadirachtin applied in a dose of 20 g a.i. ha⁻¹ against Brassica pod midge (Dasineura brassicae), has been monitored for 4 years. The biological efficiency of BI was compared with the efficiency of some synthetic insecticides. It was ascertained that BI was very efficient in decreasing the number of damaged oilseed rape pods (ranging from 56.5 to 85.9% compared to untreated plants) and its efficiency was comparable with synthetic insecticides based on Chloronicotinyl (Thiacloprid) and Neonicotinoid (Acetamiprid). BI’s efficiency was, in some years, even significantly higher compared to pyrethroid (λ-cyhalothrin). The high biological efficiency of azadirachtin that we ascertained was significantly reflected in increasing potential crop yields. The yield increase of azadirachtin ranged between 9.3 and 19.4% compared to the control sample. Azadirachtin showed the highest yield for the whole time of experimentation, and in some years the yield increase was significantly higher compared to some synthetic agents.     

Production of <Emphasis Type="Italic">Pinus halepensis</Emphasis> seedlings inoculated with the edible fungus <Emphasis Type="Italic">Lactarius deliciosus</Emphasis> under nursery conditions

Inoculation with edible fungi bestows an added value on mycorrhized plants since production of mushrooms as a secondary crop can be an economically-valuable resource in forestry management. In order to establish a protocol for controlled mycorrhization of Pinus halepensis with the edible fungus Lactarius deliciosus, several factors such as fertilisation (35, 60 or 120 mg total N and 13.5, 27 or 54 mg total P), potting substrate (sphagnum peat or sphagnum peat/vermiculite) and fungal inoculum (mycelial slurry produced in a bioreactor, alginate beads or peat/vermiculite) were assessed. The most effective inoculum was mycelial slurry at a dose of 10 ml/plant. The two potting substrates assessed were compatible with mycorrhiza formation. The greatest number of mycorrhized seedlings was obtained with a moderate level of N (35 mg/plant) or P fertilisation (27 mg/plant). Inoculation did not produce a consistent growth effect on P. halepensis, but seedlings met the quality requirements, making them suitable for planting out.