Copper-Adapted Suillus luteus, a Symbiotic Solution for Pines Colonizing Cu Mine Spoils

Natural populations thriving in heavy-metal-contaminated ecosystems are often subjected to selective pressures for increased resistance to toxic metals. In the present study we describe a population of the ectomycorrhizal fungus Suillus luteus that colonized a toxic Cu mine spoil in Norway. We hypothesized that this population had developed adaptive Cu tolerance and was able to protect pine trees against Cu toxicity. We also tested for the existence of cotolerance to Cu and Zn in S. luteus. Isolates from Cu-polluted, Zn-polluted, and nonpolluted sites were grown in vitro on Cu- or Zn-supplemented medium. The Cu mine isolates exhibited high Cu tolerance, whereas the Zn-tolerant isolates were shown to be Cu sensitive, and vice versa. This indicates the evolution of metal-specific tolerance mechanisms is strongly triggered by the pollution in the local environment. Cotolerance does not occur in the S. luteus isolates studied. In a dose-response experiment, the Cu sensitivity of nonmycorrhizal Pinus sylvestris seedlings was compared to the sensitivity of mycorrhizal seedlings colonized either by a Cu-sensitive or Cu-tolerant S. luteus isolate. In nonmycorrhizal plants and plants colonized by the Cu-sensitive isolate, root growth and nutrient uptake were strongly inhibited under Cu stress conditions. In contrast, plants colonized by the Cu-tolerant isolate were hardly affected. The Cu-adapted S. luteus isolate provided excellent insurance against Cu toxicity in pine seedlings exposed to elevated Cu levels. Such a metal-adapted Suillus-Pinus combination might be suitable for large-scale land reclamation at phytotoxic metalliferous and industrial sites.     

Increased levels of peroxisomal active oxygen-related enzymes in copper-tolerant pea plants

The effect in vivo of high nutrient levels of copper (240 micromolar) on the activity of different metalloenzymes containing Cu, Mn, Fe, and Zn, distributed in chloroplasts, peroxisomes, and mitochondria, was studied in leaves of two varieties of Pisum sativum L. plants with different sensitivity to copper. The metalloenzymes studied were: cytochrome c oxidase, Mn-superoxide dismutase (Mn-SOD) and Cu,Zn-superoxide dismutase I (Cu,Zn-SOD I), for mitochondria; catalase and Mn-SOD, for peroxisomes; and isozyme Cu,Zn-SOD II for chloroplasts. The activity of mitochondrial SOD isozymes (Mn-SOD and Cu,Zn-SOD I) was very similar in Cu-tolerant and Cu-sensitive plants, whereas cytochrome c oxidase was lower in Cu-sensitive plants. Chloroplastid Cu,Zn-SOD activity was the same in the two plant varieties. In contrast, the peroxisomal Mn-SOD activity was considerably higher in Cu-tolerant than in Cu-sensitive plants, and the activity of catalase was also increased in peroxisomes of Cu-tolerant plants. The higher activities of these peroxisomal active oxygen-related enzymes in Cu-tolerant plants suggest the involvement of reactive oxygen intermediates (O₂⁻, OH) in the mechanism of Cu lethality, and also imply a function for peroxisomal Mn-SOD in the molecular mechanisms of plant tolerance to Cu in Pisum sativum L.     

Zn pollution counteracts Cd toxicity in metal-tolerant ectomycorrhizal fungi and their host plant, Pinus sylvestris

Adaptive Zn and Cd tolerance have evolved in populations of the ectomycorrhizal fungus Suillus luteus. When exposed to high concentrations of both metals in vitro, a one-sided antagonism was apparent in the Zn- and Cd-tolerant isolates. Addition of high Zn concentrations restored growth of Cd-stressed isolates, but not vice versa. The antagonistic effect was not detected in a S. luteus isolate from non-contaminated land and in Paxillus involutus. The fungi were inoculated on pine seedlings and subsequently exposed to ecologically relevant Zn and Cd concentrations in single and mixed treatments. The applied doses severely reduced nutrient acquisition of non-mycorrhizal pines and pines inoculated with metal-sensitive S. luteus. Highest translocation of Zn and Cd to shoots occurred in the same plants. Seedlings inoculated with fungi collected from the polluted site reduced metal transfer to their host and maintained nutrient acquisition under high metal exposure. The isolate showing highest tolerance in vitro also offered best protection in symbiosis. The antagonistic effect of high Zn on Cd toxicity was confirmed in the plant experiment. The results indicate that a Zn- and Cd-polluted soil has selected ectomycorrhizal fungi that are able to survive and protect their phytobiont from nutrient starvation and excessive metal uptake.     

INFLUENCE OF PHOSPHORUS NUTRITION ON COPPER TOXICITY TO THREE STRAINS OF SCENEDESMUS ACUTUS (CHLOROPHYCEAE)1

Three strains of Scenedesmus acutus f. alternans Hortobagyi with markedly different sensitivities to copper were examined to determine the relative importance of cellular polyphosphate content on acute copper toxicity to photosynthesis. By manipulating the phosphate concentration in semicontinuous cultures, the response of each strain was assessed at three cellular phosphorus states: P-loaded, P-sufficient, and P-deficient. The results demonstrated the importance of cellular polyphosphate content in reducing the toxic effect of copper on photosynthesis; the greater the cellular P content, the less inhibition of photosynthesis occurred during copper exposure. This relationship was evident for both Cu-tolerant strains (XCu and B-4) and a Cu-sensitive strain (X72). The ranges of response to 9.9 μM Cu (measured as the percentage of control rate of photosynthesis within strains) were, from P-loaded to P-deficient cells, X72, 78–54%; XCu, 95–77%; and B-4, 99–94%. The data suggest that polyphosphate plays a passive role in protecting cells from copper; however, with respect to the mechanism of Cu tolerance, polyphosphate appeared to be relatively unimportant because the sensitivity of the Cu-tolerant strains showed less dependence on cellular polyphosphate than did the Cu-sensitive strain.     

Genetic variation and heavy metal tolerance in the ectomycorrhizal basidiomycete Suillus luteus

Twenty-one isolates of the ectomycorrhizal fungus Suillus luteus were screened for their tolerance to the heavy metals Zn, Cd, Cu and Ni, measured as inhibition of radial growth and biomass production. Two populations from even-aged pine stands were investigated: 10 isolates were obtained from an area polluted with high levels of Zn, Cd and Cu, and 11 isolates were obtained from a control population located in a nearby unpolluted area. RFLP patterns of the internal transcribed spacer region of the isolates confirmed the morphological identification of the carpophores. All isolates were maintained on basic medium without elevated metals to avoid phenotypically acquired metal tolerance. The in vitro Zn and Cd tolerance of the S. luteus isolates from the polluted habitat were significantly higher than the tolerances measured in the isolates from the nonpolluted site. This observation suggests that the elevated soil metal concentrations might be responsible for the evolution of adaptive Zn and Cd tolerance. Tolerance was maintained in an isolate not exposed to elevated metals for 3 yr. The two S. luteus populations did not differ in tolerance to Cu and Ni. The mechanisms for the adaptive Zn and Cd tolerance are not identical as there was no correlation between response to the two metals; the most Zn-tolerant isolate was the most sensitive for Cd in the metal-tolerant population. Zinc did not accumulate in basidiocarp tissue, whereas Cd levels in basidiocarps were significantly higher in the population on the polluted site. Inter-simple sequence-repeat fingerprints showed that 90% of the isolates were from different individuals. The genetic variation in the population from the unpolluted site was considerably larger than that observed at the polluted site.     

Growth and nutrient uptake of sorghum cultivated with vesicular-arbuscular mycorrhiza isolates at varying pH

This study was conducted to determine the effects of different pH regimes on root colonization with four vesicular-arbuscular mycorrhiza (VAM) isolates, and VAM effects on host plant growth and nutrient uptake. Sorghum [Sorghum bicolor (L.) Moench] was grown at pH 4.0, 5.0, 6.0 and 7.0 (±0.1) in hydroponic sand culture with the VAM isolates Glomus etunicatum UT316 (isolate E), G. intraradices UT143 (isolate I), G. intraradices UT126 (isolate B), and an unknown Glomus isolate with no INVAM number (isolate A). Colonization of roots with the different VAM isolates varied differentially with pH. As pH increased, root colonization increased with isolates B and E, remained unchanged with isolate I, and was low at pH 4.0 and high at pH 5.0, 6.0, and 7.0 with isolate A. Isolates E and I were more effective than isolates A and B in promoting plant growth irrespective of pH. Root colonization with VAM appeared to be independent of dry matter yields or dry matter yield responsiveness (dry matter produced by VAM compared to nonmycorrhizal plants). Dry matter yield responsiveness values were higher in plants whose roots were colonized with isolates E and I than with isolates A and B. Shoot P concentrations were lower in plants colonized with isolates E and I than with isolates A and B or nonmycorrhizal plants. This was probably due to the dilution effect of the higher dry matter yields. Neither the VAM isolate nor pH had an effect on shoot Ca, Mg, Zn, Cu, and Mn concentrations, while the VAM isolate affected not only P but also S, K, and Fe concentrations. The pH x VAM interaction was significant for shoot K, Mg, and Cu concentrations.     

Zinc-tolerant Suillus bovinus improves growth of Zn-exposed Pinus sylvestris seedlings

Scots pine (Pinus sylvestris L.) seedlings inoculated or not (NM) by a Zn-sensitive or a Zn-tolerant isolate of the ectomycorrhizal fungus Suillus bovinus (L. Fr.) Roussel were exposed to 0.1 or 150 μM Zn²⁺ for 9 months. We hypothesized that inoculation with a Zn-tolerant S. bovinus isolate should result in added Zn resistance of the host plant. Plant and fungal growth as well as nutrient profiles and photosynthetic pigments in pine needles were quantified. In NM plants and in plants colonized by the Zn-sensitive isolate, plant growth, N, P, Mg and Fe assimilation were strongly inhibited under Zn stress and concurred with significantly reduced chlorophyll concentrations. In contrast, plants colonized by the Zn-tolerant isolate grew much better and remained physiologically healthier when exposed to elevated Zn. These results provide further evidence for the important role metal-adapted mycorrhizal fungi play as an effective biological barrier against metal toxicity in trees.     

Copper-induced oxidative damage and enhanced antioxidant defenses in the root apex of maize cultivars differing in Cu tolerance

The root apex is highly sensitive to many soil-derived stress factors. Copper (Cu), as a Fenton-type metal, may cause severe oxidative damage in plants at toxic concentrations. The aim of this study was to establish whether the apex is the primary site of Cu-induced oxidative stress and if so, whether there is a site-specific change in antioxidant defenses that can contribute to varietal differences in Cu tolerance. For this purposes, the influence of Cu excess on cell integrity and antioxidant defenses was investigated in two maize cultivars differing in Cu tolerance, Cu-tolerant cv. Oropesa and the Cu-sensitive cv. Orense. Three root zones were considered: 0–5 mm from the root apex (including root cap, meristem and transition zone), 5–10 mm (elongation zone) and 10–15 mm (maturation zone). The 24-h exposure to nominally 2 or 5 μM Cu (pCu7 or 6) confirmed the cultivar differences in Cu tolerance. Both cell membrane integrity, especially at the transition zone in the apex, and root elongation were considerably less damaged by elevated Cu in cv. Oropesa than in cv. Orense. Root tips of both cultivars accumulated similar Cu levels (analyzed after desorption of apoplastic Cu), but 5 μM Cu induced a higher increase of SOD activity (EC 1.15.1.1) in the 0–5 mm root tip region in Oropesa than in Orense. We conclude that this apical root tip zone is the most Cu-sensitive root part, but that the better performance of cv. Oropesa is not due to greater exclusion of Cu from the root apex. Further, the local increase of SOD activity in the root apex (0–5 mm) contributed to the maintenance of cell membrane integrity in the Cu-tolerant cv. Oropesa.     

Proteomic analysis of copper stress responses in the roots of two rice (Oryza sativa L.) varieties differing in Cu tolerance

Background and aims

Copper (Cu) is an essential micronutrient required for growth and development of plants. However, excess Cu is toxic to plants. To understand the mechanisms involved in copper stress response, a proteomic approach was used to investigate the differences in Cu stress-induced protein expression between a Cu-tolerant variety (B1139) and a Cu-sensitive one (B1195) of rice.

Methods

Rice seedlings were exposed to 8 μM Cu for 3 days, with plants grown in the normal nutrient solution containing 0.32 μM Cu serving as the control. Proteins were extracted from the roots and separated by two-dimensional PAGE. Thirty four proteins were identified using MALDI-TOF mass spectrometry.

Results

Thirty-four protein spots were found to be differently expressed in the Cu-stressed roots in at least one variety of rice, including those involved in antioxidative defense, redox regulation, stress response, sulfur and glutathione (GSH) metabolism, carbohydrate metabolism, signal transduction, and some other proteins with various functions. Nine proteins, including putative cysteine synthase, probable serine acetyltransferase 3, L-ascorbate peroxidase 1, putative glutathione S-transferase 2, and thioredoxin-like 3-3, exhibited a greater increase in response to Cu stress in the Cu-tolerant variety B1139 compared with the Cu–sensitive variety B1195.

Conclusion

The majority of the proteins showing differential expression in response to Cu exposure are involved in the redox regulation, and sulfur and GSH metabolism, suggesting that these proteins, together with antioxidant enzymes, play an important role in the detoxification of excess Cu and maintaining cellular homeostasis.     

Copper-binding proteins and copper tolerance in Pisam sativum L.

The effect of high nutrient levels of copper on the low-molecular-weight copper-proteins of leaves from plants of two cultivars of Pisum sativum L., with different sensitivity to copper, was investigated. Gel-filtration chromatography of leaf extracts from Cu-tolerant and Cu-sensitive plants grown with 1 M Cu(II), showed the presence of only two copper peaks (I and II), but growth of plants with 240 M Cu(II) induced two additional copper fractions (III and IV). Fractions II and III were purified by solvent extraction, gel-filtration and ion-exchange chromatography, and their molecular weights, subunit sizes, absorption spectra, metalprotein stoichiometry and amino-acid contents were determined. Fraction II was a polypeptide of M_r 15000 composed of a single chain. The purification of fraction III produced a copper-containing fraction (III-1) of M_r 3700, and a copper-protein (III-2) with an M_r, by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, of 66000. The metal contents of fractions III-1 and III-2 were higher in Cu-tolerant than in Cu-sensitive plants. On the basis of amino-acid analyses, fraction III-1 appeared to be complexes of Cu(II)-poly-isoleucine and Cu(II)-poly-leucine. The results rule out the existence, in pea leaves, of any protein similar to either animal metallothioneins or to any of the low-molecularweight metal-binding proteins or peptides described in other plants and reported to be involved in metal tolerance. In the mechanism of copper tolerance at the leaf level, fractions III-1 (M_r 3700), III-2 (M_r 66000), and IV (M_r 2000) appear to have a role, fraction IV being specifically induced in the tolerant cultivar by Cu(II). Fractions III-1 and III-2 could participate in a different mechanism, adaptive in character, involving an enhanced capacity to bind copper in Cu-tolerant plants.Abbreviations DEAE diethylaminoethyl - M_r relative molecular mass - SDS sodium dodecyl sulfate - PAGE polyacrylamide-gel electrophoresis J.M. Palma was the recipient of a research fellowship from the Caja General de Ahorros y Monte de Piedad de Granada and CSIC. We are grateful to Dr. J. Moreno-Carretero, R + D Department, UNIASA, Granada, for conducting the amino-acid analyses. This work was supported by grant 603/275 from CAICYT-CSIC (Spain).     

Copper and cadmium tolerance, uptake and effect on chloroplast ultrastructure. Studies on Salix purpurea and Phragmites australis

We have compared the effect of toxic Cu and Cd concentrations on growth, metal accumulation, and chloroplast ultrastructure of willow (Salix purpurea L.) and reed [Phragmites australis (Cav.) Trin. ex Steud.]. After a 10-day treatment, both species have tolerated to some extent the lowest concentration of both metals; however, plant growth was strongly reduced at the highest Cu and Cd concentrations. These plants could be described as Cu-tolerant at the lowest concentration tested, showing a higher tolerance index in reed than in willow; in contrast, willow exhibited higher tolerance against Cd. Both plants appeared to be moderate root accumulators of Cu and Cd. Ultrastructural studies revealed special features that can provide some protection against heavy metals stress, such as ferritin aggregates in the stroma. In addition, Cu and Cd induced distortion of thylakoids, reduction of grana stacks, as well as an increased number and size of plastoglobuli and peripheral vesicles.     

Chlorophyll fluorescence and photoacoustic characteristics in relationship to changes in chlorophyll and Ca2+ content of a Cu-tolerant Silene compacta ecotype under Cu treatment

The effect of Cu toxicity on photosynthetic function, chlorophyll and Ca²⁺ content of Cu-tolerant Silene compacta plants grown in nutrient solution was studied. Since, in plants grown under 8 μ M Cu, the chlorophyll and Ca²⁺ concentration as well as the photosystem II (PSII) photochemistry were increased, compared to the control, the development of an adaptive mechanism of the Cu-tolerant ecotype of S. compacta to 8 μ M Cu is suggested. Increased Cu tolerance of the S. compacta ecotype reflects modulation of the photosynthetic apparatus to optimize photosynthesis. However, exposure of plants to 160 μ M Cu resulted in a marked increase of the fraction of closed PSII centres and decreased quantum yield of PSII electron transport (Φ_PSU) which was accompanied by a significant decline of relative quantum yield for O₂ evolution (A_ox/A_pt). The concentration of chlorophyll and Ca²⁺ in leaves also decreased significantly under 160 μ M Cu treatment. Photochemical quenching (q_p) displayed a reduction as a result of perturbation of the photosynthetic electron transfer chain, while non-photochemical quenching (q_N) increased. High Cu treatment reduced photosynthetic productivity of S. compacta plants which can be attributed, in part, to pertubation of photosynthetic process and photosynthetic pigments as well as to Ca²⁺ loss.     

Differential invertase activity and root growth between Cu-tolerant and non-tolerant populations in Kummerowia stipulacea under Cu stress and nutrient deficiency

There has been no study on key enzymes in sucrose cleavage in metallophyte plants so far, which may be crucial for the plants’ root growth and heavy-metal tolerance maintenance. Here, we tested the hypothesis that the roots of copper tolerant plants should manifest a higher activity of acid invertases that are rate-limiting in sucrose catabolism than non-tolerant plants both for supporting growth and for their maintaining tolerance under Cu stress. Two populations of Kummerowia stipulacea, one from an ancient waste heap at a Cu mine, and the other from a non-contaminated site, were used in the experiments. The plants were grown in 1/2-fold (control) or 1/20-fold (nutrient deficiency) Hoagland’ solution, with (Cu stress) or without (control) 10 μmol/L Cu²⁺. Plants from the mine proved to be of Cu tolerance. Cu exposure had a stronger inhibition on root growth and thus resulting in a lower root/shoot ratio in the plants of non-mine population compared to the mine population. Cu exposure showed a stronger inhibition of acid invertase activity of Cu non-tolerant plants than Cu-tolerant plants, while neutral/alkaline invertase was insensitive to Cu. A positive correlation between the activity of acid invertases and the root growth and root/shoot ratio was observed. The results indicated an important role of acid invertases in governing root growth and root/shoot biomass allocation in the plants of mine population. The results also suggested that the higher activities in acid invertases of mine population plants might at least partly associate with the plants’ Cu tolerance, and their higher activities in acid invertases in turn played an role in maintenance of the Cu tolerance by supplying carbon and energy for tolerance mechanisms. In addition, the results showed evidence that neutral/alkaline invertase might play a role in compensating for the depression in sucrose catabolism due to Cu-induced inhibition in acid invertases.     

Response of ectomycorrhizalPinus banksiana andPicea glauca to heavy metals in soil

Pinus banksiana andPicea glauca inoculated or not with the ectomycorrhizal fungusSuillus luteus were grown in a sandy loam soil containing a range of Cd, Cu, Ni, Pb and Zn concentrations. Ectomycorrhizal colonization rates were significantly reduced on Pinus and Picea seedlings by the heavy metals, particularly Cd and Ni. Needle tissue metal concentrations were lower in ectomycorrhizal seedlings at low soil metal concentrations. However, at higher soil concentrations, heavy metal concentrations of needle tissue were similar in ectomycorrhizal and nonmycorrhizal plants. The growth of nonmycorrhizal seedlings exposed to heavy metals was reduced compared to those inoculated withSuillus luteus. Apparently ectomycorrhizal colonization can protect Pinus and Picea seedlings from heavy metal toxicity at low or intermediate soil concentrations of Cd, Cu, Ni, Pb and Zn.     

IV. Variation in fungicide tolerance: Comparisons between isolates of Erysiphe graminis f. sp. hordei tolerant and sensitive to the fungicide tridemorph

Forty-five single pustule isolates of barley powdery mildew taken at random from tridemorph treated and untreated plants were maintained in culture on leaf segments of barley cv. Golden Promise placed on agar containing benzimidazole. In repeated assays for tridemorph tolerance over a 10 month period, tolerance was found in 25 of 36 isolates from treated plants, and remained stable in all but four of the isolates wherein tolerance was lost after varying lengths of time. A negative correlation was found between pathogenicity and tolerance. The germination of tolerant isolates was less depressed than the sensitive on treated seedlings, but no differences between isolates were found on untreated seedlings. Colony growth of all isolates was slower on treated than untreated seedlings. On plants of several cultivars kept in growth rooms, mildew progressed more rapidly on treated barley when tolerant isolates were used as inoculum. Visible disease symptoms spread most rapidly on isolated, untreated plants, and most slowly on isolated treated plants, irrespective of the isolate. Disease on treated plants progressed more rapidly when untreated plants were adjacent, suggesting that interactions take place between pathogen populations on treated and untreated plants.     

Field response of wheat to arbuscular mycorrhizal fungi and drought stress

Mycorrhizal plants often have greater tolerance to drought than nonmycorrhizal plants. This study was conducted to determine the effects of arbuscular mycorrhizal (AM) fungi inoculation on growth, grain yield and mineral acquisition of two winter wheat (Triticum aestivum L.) cultivars grown in the field under well-watered and water-stressed conditions. Wheat seeds were planted in furrows after treatment with or without the AM fungi Glomus mosseae or G. etunicatum. Roots were sampled at four growth stages (leaf, tillering, heading and grain-filling) to quantify AM fungi. There was negligible AM fungi colonization during winter months following seeding (leaf sampling in February), when soil temperature was low. During the spring, AM fungi colonization increased gradually. Mycorrhizal colonization was higher in well-watered plants colonized with AM fungi isolates than water-stressed plants. Plants inoculated with G. etunicatum generally had higher colonization than plants colonized with G. mosseae under both soil moisture conditions. Biomass and grain yields were higher in mycorrhizal than nonmycorrhizal plots irrespective of soil moisture, and G. etunicatum inoculated plants generally had higher biomass and grain yields than those colonized by G. mosseae under either soil moisture condition. The mycorrhizal plants had higher shoot P and Fe concentrations than nonmycorrhizal plants at all samplings regardless of soil moisture conditions. The improved growth, yield and nutrient uptake in wheat plants reported here demonstrate the potential of mycorrhizal inoculation to reduce the effects of drought stress on wheat grown under field conditions in semiarid areas of the world.     

Apatite as a P source in mycorrhizal and non-mycorrhizal Pinus sylvestris seedlings

The objectives of the study are firstly to test the ability of ectomycorrhizal pine seedlings to use apatite as a P source in comparison with non-mycorrhizal pine seedlings and secondly, to determine if there is a relation between exudation of organic acids and the ability to use apatite as a P source. Non-mycorrhizal Pinus sylvestris (L.) seedlings and seedlings ectomycorrhizal with 4 different isolates of ectomycorrhizal fungi were grown for 220 days in sand/peat filled pots with apatite (Ca₅(F,OH)(PO₄)₃) as the sole P source. In an additional experiment, non-mycorrhizal Pinus sylvestris (L.) seedlings and seedlings ectomycorrhizal with 2 different isolates of ectomycorrhizal fungi were grown without any P source for 250 days. All other nutrients were supplied in a balanced nutrient solution.Ectomycorrhizal seedlings grew less than non-mycorrhizal seedlings but ectomycorrhizal seedlings produced a large external mycelium not included in the biomass estimates. All seedlings in the present study had low shoot:root ratios compared to seedlings growing under optimal conditions. All seedlings grown with apatite as P source had higher foliar P concentrations (0.71–2.11 mg/g) than seedlings growing without any P source (0.57–0.75 mg/g) indicating a significant ability to use apatite as a P source. Seedlings colonized by Suillus variegatus and Paxillus involutus had higher concentrations and total contents of P in shoots compared with non-mycorrhizal seedlings, indicating significant improvement of P uptake by these fungi in comparison with non-mycorrhizal seedlings or seedlings colonized Piloderma croceum.No clear relationship between exudation of organic acids and uptake of P was found. Seedlings colonized by S. variegatus reduced the pH of the soil more than seedlings colonized by P. involutus or non-mycorrhizal seedlings. It is suggested that S. variegatus colonization improves the P uptake by reducing the pH of the soil while P. involutus improves P uptake by having a greater ability to absorb dissolved phosphate than non-mycorrhizal roots or roots colonized by the other fungi used in the study.     

Comparative Studies between Portuguese and Japanese Isolates of the Pinewood Nematode, Bursaphelenchus xylophilus

Comparative studies between Portuguese (T and HF) and Japanese (S10, T4, C14-5 and OKD-1) isolates of the pinewood nematode Bursaphelenchus xylophilus have been made in order to provide information to better understand the possible origin of the Portuguese isolates, recently introduced in the European Union. The main comparative aspects investigated were pathogenicity (seedling mortality ratio), sexual compatibility, and DNA sequences of the rDNA region. Four-year-old Japanese black pine (Pinus thunbergii) seedlings were used as host plants for pathogenicity tests. The Portuguese isolates, and in particular isolate “T,” propagated in higher numbers than the Japanese isolates within pine seedlings. All combinations of crossings produced viable progeny, with higher numbers obtained when crossings were made between Japanese and Portuguese isolates, a possible situation of heterosis and/or inbreeding depression. Reciprocal crossings yielded different values, which may reflect a sex effect (maternal inheritance, mtDNA). Regarding DNA sequencing, both Portuguese isolates displayed nearly identical ITS 1, ITS2, and 5.8S rDNA base sequences as the Japanese isolates. Although biologically very similar, and possibly reflecting a common origin, the Portuguese isolates may present a serious threat to Japanese black pine, due to their higher virulence.     

Colonization of Metarhizium anisopliae on the surface of pine tree logs: A promising biocontrol strategy for the Japanese pine sawyer,Monochamus alternatus

We investigated the colonization potential of five Metarhizium anisopliae isolates on pine tree surfaces under laboratory conditions, determined the influence of the pine bark extract on fungal growth and evaluated the insecticidal activity following colonization on the Japanese pine sawyer. Finally, the effect of colonization on adults pine sawyer was evaluated using the top three performing isolates (JEF-197, JEF-271 and JEF-279) under laboratory and field conditions. As a result, isolate JEF-197 showed the highest conidial production on the pine surfaces, and five isolates, including JEF-197, showed higher hyphal growth on autoclaved pine bark extract agar, compared to a water agar. Pine bark treated with the isolates showed 40–70 % mortality of adults pine sawyer. Under mimicked overwintering conditions, in the JEF-197 treatment group, 40 % of the inserted larvae became adults and all were dead after 59 d. In a field test, colonized isolate JEF-197 also showed 37 % insecticidal activity against emerged adults from the pine logs as overwintering sites. This work suggests that M. anisopliae isolate JEF-197 possibly colonized the pine surface and application of a conidial suspension on the pine logs as overwintering sites could be an effective strategy to control the pine sawyer.