Ectomycorrhizal fungal community structure in a young orchard of grafted and ungrafted hybrid chestnut saplings.

Ectomycorrhizal (ECM) fungal community of the European chestnut has been poorly investigated, and mostly by sporocarp sampling. We proposed the study of the ECM fungal community of 2-year-old chestnut hybrids Castanea × coudercii (Castanea sativa × Castanea crenata) using molecular approaches. By using the chestnut hybrid clones 111 and 125, we assessed the impact of grafting on ECM colonization rate, species diversity, and fungal community composition. The clone type did not have an impact on the studied variables; however, grafting significantly influenced ECM colonization rate in clone 111. Species diversity and richness did not vary between the experimental groups. Grafted and ungrafted plants of clone 111 had a different ECM fungal species composition. Sequence data from ITS regions of rDNA revealed the presence of 9 orders, 15 families, 19 genera, and 27 species of ECM fungi, most of them generalist, early-stage species. Thirteen new taxa were described in association with chestnuts. The basidiomycetes Agaricales (13 taxa) and Boletales (11 taxa) represented 36% and 31%, of the total sampled ECM fungal taxa, respectively. Scleroderma citrinum, S. areolatum, and S. polyrhizum (Boletales) were found in 86% of the trees and represented 39% of total ECM root tips. The ascomycete Cenococcum geophilum (Mytilinidiales) was found in 80% of the trees but accounted only for 6% of the colonized root tips. These results could help to unveil the impact of grafting on fungal symbionts, improving management of chestnut agro-ecosystems and production of edible fungal species.

Bottom-up and top-down herbivore regulation mediated by glucosinolates in Brassica oleracea var. acephala.

Quantitative differences in plant defence metabolites, such as glucosinolates, may directly affect herbivore preference and performance, and indirectly affect natural enemy pressure. By assessing insect abundance and leaf damage rate, we studied the responses of insect herbivores to six genotypes of Brassica oleracea var. acephala, selected from the same cultivar for having high or low foliar content of sinigrin, glucoiberin and glucobrassicin. We also investigated whether the natural parasitism rate was affected by glucosinolates. Finally, we assessed the relative importance of plant chemistry (bottom-up control) and natural enemy performance (top-down control) in shaping insect abundance, the ratio of generalist/specialist herbivores and levels of leaf damage. We found that high sinigrin content decreased the abundance of the generalist Mamestra brassicae (Lepidoptera, Noctuidae) and the specialist Plutella xylostella (Lepidoptera, Yponomeutidae), but increased the load of the specialist Eurydema ornatum (Hemiptera, Pentatomidae). Plants with high sinigrin content suffered less leaf injury. The specialist Brevicoryne brassicae (Hemiptera, Aphididae) increased in plants with low glucobrassicin content, whereas the specialists Pieris rapae (Lepidoptera, Pieridae), Aleyrodes brassicae (Hemiptera, Aleyrodidae) and Phyllotreta cruciferae (Coleoptera, Chrysomelidae) were not affected by the plant genotype. Parasitism rates of M. brassicae larvae and E. ornatum eggs were affected by plant genotype. The ratio of generalist/specialist herbivores was positively correlated with parasitism rate. Although both top-down and bottom-up forces were seen to be contributing, the key factor in shaping both herbivore performance and parasitism rate was the glucosinolate concentration, which highlights the impact of bottom-up forces on the trophic cascades in crop habitats.

Effects of selected metal oxide nanoparticles on Artemia salina larvae: evaluation of mortality and behavioural and biochemical responses.

The aim was to investigate the toxicity of selected metal oxide nanoparticles (MO-NPs) on the brine shrimp Artemia salina, by evaluating mortality and behavioural and biochemical responses. Larvae were exposed to tin(IV) oxide (stannic oxide (SnO2)), cerium(IV) oxide (CeO2) and iron(II, III) oxide (Fe3O4) NPs for 48 h in seawater, with MO-NP suspensions from 0.01 to 1.0 mg/mL. Mortality and behavioural responses (swimming speed alteration) and enzymatic activities of cholinesterase, glutathione-S-transferase and catalase were evaluated. Although the MO-NPs did not induce any mortality of the larvae, they caused changes in behavioural and biochemical responses. Swimming speed significantly decreased in larvae exposed to CeO2 NPs. Cholinesterase and glutathione-S-transferase activities were significantly inhibited in larvae exposed to SnO2 NPs, whereas cholinesterase activity significantly increased after CeO2 NP and Fe3O4 NP exposure. Catalase activity significantly increased in larvae exposed to Fe3O4 NPs. In conclusion, swimming alteration and cholinesterase activity represent valid endpoints for MO-NP exposure, while glutathione-S-transferase and catalase activities appear to be NP-specific.

Defoliation negatively affects plant growth and the ectomycorrhizal community of Pinus pinaster in Spain.

In this work, by artificially reproducing severe (75%) and moderate (25%) defoliation on maritime pines Pinus pinaster in NW Spain, we investigated, under natural conditions, the consequences of foliage loss on reproduction, abundance, diversity and richness of the fungal symbionts growing belowground and aboveground. The effect of defoliation on tree growth was also assessed. Mature needles were clipped during April 2007 and 2008. Root samples were collected in June-July 2007 and 2008. Collection of sporocarps was performed weekly from April 2007 to April 2009. Taxonomic identity of ectomycorrhizal fungi was assessed by using the internal transcribed spacer (ITS) regions of rDNA through the polymerase chain reaction (PCR) method, subsequent direct sequencing and BLAST search. Ectomycorrhizal colonization was significantly reduced (from 54 to 42%) in 2008 by 75% defoliation, accompanied with a decline in species richness and diversity. On the other hand, sporocarp abundance, richness and diversity were not affected by foliage loss. Some ECM fungal symbionts, which are assumed to have a higher carbon cost according to the morphotypes structure, were reduced due to severe (75%) defoliation. Furthermore, 75% foliage loss consistently depressed tree growth, which in turn affected the ectomycorrhizal growth pattern. Defoliation impact on ECM symbionts largely depends on the percentage of foliage removal and on the number of defoliation bouts. Severe defoliation (75%) in the short term (2 years) changed the composition of the ECM community likely because root biomass would be adjusted to lower levels in parallel with the depletion of the aboveground plant biomass, which probably promoted the competition among mycorrhizal types for host resources. The persistence of fungal biomass in mycorrhizal roots would be crucial for nutrient up-take and recovery from defoliation stress of the host plants.

Context dependency, co-introductions, novel mutualisms, and host shifts shaped the ectomycorrhizal fungal communities of the alien tree Eucalyptus globulus.

The identity and relevance of the ectomycorrhizal (ECM) fungal partners of Eucalyptus globulus was investigated in NW Spain, to detect which symbionts mainly support its invasiveness. Root tips of E. globulus and of three common native plant species (Quercus robur, Pinus pinaster and Halimium lasianthum) were collected in eucalypt plantations, Q. robur forests, P. pinaster plantations and shrublands. Fungal taxonomical identity was ascertained by use of rDNA and direct sequencing. We studied diversity, composition and colonization rate of the ECM fungal communities of E. globulus to determine if fungal assemblages are host specific (i.e. similar in different habitats) or more dependent on the neighbourhood context. We also identified the type of associations formed (i.e. co-introductions, familiar or novel associations). Twenty-six ECM taxa were associated with E. globulus. Most of them engaged in novel associations with eucalypts, whereas only three fungal species were co-introduced Australian aliens. Eucalypt fungal richness, diversity and colonization rate differed between habitats, being higher in native oak forests, whereas in shrublands E. globulus showed the lowest colonization rate and diversity. The Australian fungus Descolea maculata dominated the eucalypt fungal assemblage and also spread to the native host plants, in all the habitats, posing the risk of further co-invasion.

Pest categorisation of the Gonipterus scutellatus species complex.

The Panel on Plant health performed a pest categorisation of the Australian Eucalyptus snout-beetle Gonipterus scutellatus (Coleoptera: Curculionidae), for the EU. G. scutellatus should be referred as the G. scutellatus species complex because it includes several cryptic species. A complete nomenclature of the species present in the EU is still pending. It is a quarantine pest listed in Annex IIB of Council Directive 2000/29/EC. Protected zones are in place in Greece and Portugal (Azores). In the EU, it has been found in Italy, France, Spain and Portugal. It only consumes Eucalyptus species leaves. The main pathways of spread are the trade of Eucalyptus timber, hitchhiking in various commodities, trade of apple fruit as well as of plants for planting or plant parts. Spread by flight is also possible. The climate of the EU protected zones is similar to that of the Member States (MS) where the G. scutellatus complex is established, and the pest's main host plants are present. The damaged trees suffer die-back and the development of epicormics shoots. Severe attacks may provoke massive amounts of tree death. Biological control by using the egg parasitoid wasp Anaphes nitens is the most effective control measure. Some species within the G. scutellatus complex are not yet present in the EU (including G. scutellatus sensu stricto) and might therefore be considered as potential union quarantine pests for the EU territory. At least two species within the G. scutellatus complex (most likely G. platensis and Gonipterus species no. 2) meet the criteria assessed by EFSA for consideration as potential protected zone quarantine pests for the territory of the protected zones: Greece and Portugal (Azores). The criteria for considering the G. scutellatus complex as a potential regulated non-quarantine pest for the EU are not met since plants for planting are not the main pathway.

Bioavailability of CeO2 and SnO2 nanoparticles evaluated by dietary uptake in the earthworm Eisenia fetida and sequential extraction of soil and feed.

The growing number of nanotechnology products on the market will inevitably lead to the release of engineered nanomaterials with potential risk to humans and environment. This study set out to investigate the exposure of soil biota to engineered nanoparticles (NPs). Cerium dioxide (CeO2 NPs) and tin dioxide nanoparticles (SnO2 NPs) were radiolabelled using neutron activation, and employed to assess the uptake and excretion kinetics in the earthworm Eisenia fetida. Through sequential extraction, NPs bioavailability in two contrasting soils and in earthworm feed was also investigated. Neither CeO2 NPs nor SnO2 NPs bioaccumulated in earthworms, and both were rapidly excreted when worms were transferred to clean soil. Low bioavailability was also indicated by low amounts of NPs recovered during extraction with non-stringent extractants. CeO2 NPs showed increasing mobility in organic soil over time (28 days), indicating that organic matter has a strong influence on the fate of CeO2 NPs in soil.

Uptake and translocation of metals and nutrients in tomato grown in soil polluted with metal oxide (CeO2, Fe3O4, SnO2, TiO2) or metallic (Ag, Co, Ni) engineered nanoparticles.

The influence of exposure to engineered nanoparticles (NPs) was studied in tomato plants, grown in a soil and peat mixture and irrigated with metal oxides (CeO2, Fe3O4, SnO2, TiO2) and metallic (Ag, Co, Ni) NPs. The morphological parameters of the tomato organs, the amount of component metals taken up by the tomato plants from NPs added to the soil and the nutrient content in different tomato organs were also investigated. The fate, transport and possible toxicity of different NPs and nutrients in tomato tissues from soils were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES). The tomato yield depended on the NPs: Fe3O4-NPs promoted the root growth, while SnO2-NP exposure reduced it (i.e. +152.6 and -63.1 % of dry matter, respectively). The NP component metal mainly accumulated in the tomato roots; however, plants treated with Ag-, Co- and Ni-NPs showed higher concentration of these elements in both above-ground and below-ground organs with respect to the untreated plants, in addition Ag-NPs also contaminated the fruits. Moreover, an imbalance of K translocation was detected in some plants exposed to Ag-, Co- and Fe3O4-NPs. The component metal concentration of soil rhizosphere polluted with NPs significantly increased compared to controls, and NPs were detected in the tissues of the tomato roots using electron microscopy (ESEM-EDS).

Characterization of juvenile maritime pine (Pinus pinaster Ait.) ectomycorrhizal fungal community using morphotyping, direct sequencing and fruitbodies sampling.

Using ectomycorrhizal root tip morphotyping (anatomical and morphological identification), molecular analysis (internal transcribed spacer region amplification and sequencing), and fruitbody sampling, we assessed diversity and composition of the ectomycorrhizal fungal community colonizing juvenile Pinus pinaster Ait. under natural conditions in NW Spain. Overall, we found 15 Basidiomycetes and two Ascomycetes. Members of the family Thelephoraceae represented up to 59.4% of the samples. The most frequent species was Tomentella sublilacina followed by Thelephora terrestris, Russula drimeia, Suillus bovinus, and Paxillus involutus, while the less frequent were Pseudotomentella tristis, Lactarius subdulcis, Russula ochroleuca, and Entoloma conferendum. From October 2007 to June 2008, we sampled 208 sporocarps belonging to seven genera and nine species: Thelephora terrestris, Paxillus involutus, Suillus bovinus, Xerocomus badius, Scleroderma verrucosum, Amanita gemmata, A. rubescens, Amanita sp., and Russula sp. The species belonging to the genus Amanita, X. badius and S. verrucosum were not found on root samples. By comparing our results with a bibliographic review of papers published from 1922 to 2006, we found five genera and six species which have not been previously reported in symbiosis with P. pinaster. This is the first time that the diversity of the ectomycorrhizal fungal community associated with P. pinaster was investigated using molecular techniques. Considering that only 38% of the genera found by sequencing were found as fruitbodies, we conclude that integrating morphotyping and sporocarps surveys with molecular analysis of ectomycorrhizas is important to documenting the ectomycorrhizal fungus community.

Superparasitism and sex ratio adjustment in a wasp parasitoid: results at variance with Local Mate Competition?

Anaphes nitens is a solitary parasitoid of the egg capsules of the Eucalyptus snout beetle, Gonipterus scutellatus. Some traits of its natural history suggest that Local Mate Competition (LMC) could account for sex ratio adjustment in this species. We tested whether males emerged early, a prerequisite for fully local mating, and investigated the occurrence and effect of superparasitism on adult size and pre-emergence mortality, factors that might influence sex ratio adjustment. We found in field-collected egg capsules that males emerged first. To investigate the effects of superparasitism on adult size, we compared the sizes of parasitoids that emerged early and late from egg capsules collected in the field, and from egg capsules parasitized and superparasitized in the laboratory. Superparasitism reduced parasitoid size, affecting females more strongly than males, and increased pre-emergence mortality. We estimated A. nitens sex ratio and parasitism rate in the field during 2 years in five localities and during 4 years in a sixth. Following LMC we expected an increase in sex ratio (proportion of males) with increasing parasitism rate (assumed to reflect parasitoid density). We found that sex ratio decreased from 0.38 when the parasitism rate was low (0-20%) to 0.21 when parasitism was high (80-100%). In contrast with field results, a laboratory experiment showed that: (1) at a low parasitism level sex ratio was clearly female biased (0.28+/-0.04), (2) at a high parasitism level sex ratio increased (0.40+/-0.07), (3) male larval survivorship was not lower than female survivorship, and (4) low-quality hosts (i.e. superparasitized) were allocated more males. We conclude that LMC cannot explain the sex ratio adjustment observed in the field, even at low parasitism rates, and alternative explications for highly female-biased sex ratios must be found. One such alternative is female-biased dispersal.