Restinga ectomycorrhizae: a work in progress.

Background: The Brazilian Atlantic Forest is one of the most biodiverse terrestrial ecoregions of the world. Among its constituents, restinga vegetation makes a particular case, acting as a buffer zone between the oceans and the forest. Covering some 80% of Brazilian coastline (over 7,300 km in length), restinga is a harsh environment where plants and fungi interact in complex ways that just now are beginning to be unveiled. Ectomycorrhizal symbiosis, in particular, plays a so far ungauged and likely underestimated role. We recently described the morpho-anatomical and molecular features of the ectomycorrhizae formed by several basidiomycetous mycobionts on the host plant Guapira opposita, but the mycorrhizal biology of restinga is still largely unexplored. Here, we report new data on the ectomycorrhizal fungal symbionts of G. opposita, based on the collection of sporomata and ectomycorrhizal root tips in restinga stands occurring in southern Brazil. Methods: To obtain a broader view of restinga mycorrhizal and ecological potential, we compiled a comprehensive and up-to-date checklist of fungal species reported or supposed to establish ectomycorrhizae on restinga-inhabiting host plants, mainly on the basis of field observations. Results: Our list comprises some 726 records, 74 of which correspond to putative ectomycorrhizal taxa specifically associated with restinga. These include several members of Boletaceae, Amanita, Tomentella/ Thelephora, Russula/ Lactifluus, and Clavulina, as well as hypogeous fungi, like the recently described Longistriata flava. Conclusions: Our survey reveals a significant diversity of the restinga ectomycorrhizal mycobiota, indicating the importance of this symbiosis for the ecological functioning of a unique yet poorly known and threatened ecosystem.

Fluorescent protein expression in the ectomycorrhizal fungus Laccaria bicolor: a plasmid toolkit for easy use of fluorescent markers in basidiomycetes.

For long time, studies on ectomycorrhiza (ECM) have been limited by inefficient expression of fluorescent proteins (FPs) in the fungal partner. To convert this situation, we have evaluated the basic requirements of FP expression in the model ECM homobasidiomycete Laccaria bicolor and established eGFP and mCherry as functional FP markers. Comparison of intron-containing and intronless FP-expression cassettes confirmed that intron-processing is indispensable for efficient FP expression in Laccaria. Nuclear FP localization was obtained via in-frame fusion of FPs between the intron-containing genomic gene sequences of Laccaria histone H2B, while cytosolic FP expression was produced by incorporating the intron-containing 5' fragment of the glyceraldehyde-3-phosphate dehydrogenase encoding gene. In addition, we have characterized the consensus Kozak sequence of strongly expressed genes in Laccaria and demonstrated its boosting effect on transgene mRNA accumulation. Based on these results, an Agrobacterium-mediated transformation compatible plasmid set was designed for easy use of FPs in Laccaria. The four cloning plasmids presented here allow fast and highly flexible construction of C-terminal in-frame fusions between the sequences of interest and the two FPs, expressed either from the endogenous gene promoter, allowing thus evaluation of the native regulation modes of the gene under study, or alternatively, from the constitutive Agaricus bisporus gpdII promoter for enhanced cellular protein localization assays. The molecular tools described here for cell-biological studies in Laccaria can also be exploited in studies of other biotrophic or saprotrophic basidiomycete species susceptible to genetic transformation.

Dual-mycorrhizal plants: their ecology and relevance.

Dual-mycorrhizal plants are capable of associating with fungi that form characteristic arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) structures. Here, we address the following questions: (1) How many dual-mycorrhizal plant species are there? (2) What are the advantages for a plant to host two, rather than one, mycorrhizal types? (3) Which factors can provoke shifts in mycorrhizal dominance (i.e. mycorrhizal switching)? We identify a large number (89 genera within 32 families) of confirmed dual-mycorrhizal plants based on observing arbuscules or coils for AM status and Hartig net or similar structures for EM status within the same plant species. We then review the possible nutritional benefits and discuss the possible mechanisms leading to net costs and benefits. Cost and benefits of dual-mycorrhizal status appear to be context dependent, particularly with respect to the life stage of the host plant. Mycorrhizal switching occurs under a wide range of abiotic and biotic factors, including soil moisture and nutrient status. The relevance of dual-mycorrhizal plants in the ecological restoration of adverse sites where plants are not carbon limited is discussed. We conclude that dual-mycorrhizal plants are underutilized in ecophysiological-based experiments, yet are powerful model plant-fungal systems to better understand mycorrhizal symbioses without confounding host effects.

Arbuscular and Ectomycorrhizal Fungi Associated with the Invasive Brazilian Pepper Tree (Schinus terebinthifolius) and Two Native Plants in South Florida.

The potential role of soil fungi in the invasion of the Brazilian pepper tree (Schinus terebinthifolius-BP) in Florida is not known; although the low biotic resistance of Florida soils is often invoked to explain the prevalence of many invasive species. To gain an initial insight into BP's mycorrhizal associations, this study examined the rhizobiome of BP and two native plants (Hamelia patens and Bidens alba) across six locations. Arbuscular mycorrhizal fungi (AMF) associated with the roots of the target plants and bulk soil was characterized by spore morphotyping. Sequence analysis of metagenomic DNA from lateral roots/rhizosphere of BP (n = 52) and a native shrub H. patens (n = 37) on the same parcel yielded other fungal associates. Overall, the total population of AMF associated with BP was about two folds greater than that of the two native plants (p = 0.0001) growing on the same site. The dominant AMF under Schinus were members of the common Glomus and Rhizophagus spp. By contrast, the most prevalent AMF in the bulk soil and rhizosphere of the two Florida native plants, Acaulospora spp (29%) was sharply diminished (9%) under BP rhizosphere. Analysis of the ITS2 sequences also showed that Schinus rhizosphere had a high relative abundance of ectomycorrhizal fungi (76.5%) compared to the native H. patens (2.6%), with the species Lactifluus hygrophoroides (Basidiomycota) being the most prevalent at 61.5% (p < 0.05). Unlike the native plants where pathogenic fungi like Phyllosticta sp., Phoma sp., and Neofusicoccum andium were present (8.1% for H. patens), only one potentially pathogenic fungal taxon was detected (3.9%) under BP. The striking disparity in the relative abundance of AMF and other fungal types between BP and the native species is quite significant. Fungal symbionts could aide plant invasion via resource-use efficiency and other poorly defined mechanisms of protection from pathogens in their invaded range. This report exposes a potentially significant but previously unappreciated fungal dimension of a complex invasion system and underscores the need to characterize these fungal symbionts, their role and mode of action during invasion; with the goal of devising measures for invasion control and ecological restoration.

Impact of alien pines on local arbuscular mycorrhizal fungal communities-evidence from two continents.

The introduction of alien plants can influence biodiversity and ecosystems. However, its consequences for soil microbial communities remain poorly understood. We addressed the impact of alien ectomycorrhizal (EcM) pines on local arbuscular mycorrhizal (AM) fungal communities in two regions with contrasting biogeographic histories: in South Africa, where no native EcM plant species are present; and in Argentina, where EcM trees occur naturally. The effect of alien pines on AM fungal communities differed between these regions. In South Africa, plantations of alien EcM pines exhibited lower AM fungal richness and significantly altered community composition, compared with native fynbos. In Argentina, the richness and composition of local AM fungal communities were similar in plantations of alien EcM pines and native forest. However, the presence of alien pines resulted in slight changes to the phylogenetic structure of root AM fungal communities in both regions. In pine clearcut areas in South Africa, the richness and composition of AM fungal communities were intermediate between the native fynbos and the alien pine plantation, which is consistent with natural regeneration of former AM fungal communities following pine removal. We conclude that the response of local AM fungal communities to alien EcM pines differs between biogeographic regions with different histories of species coexistence.

New species of Tomentella (Thelephorales) from the Patagonian Andes forests.

The genus Tomentella forms abundant ectomycorrhizae in coniferous and deciduous forests worldwide. Molecular identification of root tips suggests undescribed species in the Nothofagus forests of Patagonia, Argentina. Tomentella tenuissima, T. pulvinulata and T. patagonica are described here as new to science based on morphological and molecular analyses. Their host range is addressed using available soil sequences. The identity of previous records of T. galzinii and T. radiosa are discussed with morphological and molecular evidence.

Biochemical and ecophysiological responses to manganese stress by ectomycorrhizal fungus Pisolithus tinctorius and in association with Eucalyptus grandis.

At relatively low concentrations, the element manganese (Mn) is essential for plant metabolism, especially for photosynthesis and as an enzyme antioxidant cofactor. However, industrial and agricultural activities have greatly increased Mn concentrations, and thereby contamination, in soils. We tested whether and how growth of Pisolithus tinctorius is influenced by Mn and glucose and compare the activities of oxidative stress enzymes as biochemical markers of Mn stress. We also compared nutrient accumulation, ecophysiology, and biochemical responses in Eucalyptus grandis which had been colonized by the ectomycorrhizal Pisolithus tinctorius with those which had not, when both were exposed to increasing Mn concentrations. In vitro experiments comprised six concentrations of Mn in three concentrations of glucose. In vivo experiments used plants colonized by Pisolithus tinctorius, or not colonized, grown with three concentrations of Mn (0, 200, and 1000 µM). We found that fungal growth and glucose concentration were correlated, but these were not influenced by Mn levels in the medium. The anti-oxidative enzymes catalase and glutathione S-transferase were both activated when the fungus was exposed to Mn. Also, mycorrhizal plants grew more and faster than non-mycorrhizal plants, whatever Mn exposure. Photosynthesis rate, intrinsic water use efficiency, and carboxylation efficiency were all inversely correlated with Mn concentration. Thus, we originally show that the ectomycorrhizal fungus provides protection for its host plants against varying and potentially toxic concentrations of Mn.

Molecular and morphological analyses confirm Rhizopogon verii as a widely distributed ectomycorrhizal false truffle in Europe, and its presence in South America.

The genus Rhizopogon includes species with hypogeous or subepigeus habit, forming ectomycorrhizae with naturally occurring or planted pines (Pinaceae). Species of the genus Rhizopogon can be distinguished easily from the other hypogeous basidiomycetes by their lacunose gleba without columella and their smooth elliptical spores; however, the limit between species is not always easy to establish. Rhizopogon luteolus, the type species of the genus, has been considered one of the species that are more abundant in Europe, as well as it has been cited in pine plantation of North and South America, different parts of Africa, Australia, and New Zealand. However, in this study, based on molecular analyses of the ITS nuclear ribosomal DNA (nrDNA) sequences (19 new sequences; 37 sequences from GenBank/UNITE, including those from type specimens), we prove that many GenBank sequences under R. luteolus were misidentified and correspond to Rhizopogon verii, a species described from Tunisia. Also, we confirm that basidiomes and ectomycorrhizae recently collected in Germany under Pinus sylvestris, as well as specimens from South of Brazil under Pinus taeda belong to R. verii. Thanks to the numerous ectomycorrhizal tips collected in Germany, a complete description of R. verii/P. sylvestris ectomycorrhiza is provided. Moreover, since in this paper the presence of R. verii in South America is here reported for the first time, a short description of basidiomes collected in Brazil, compared with collections located in different European herbaria, is included.

Eucalyptus grandis Hill ex Maiden inoculated with Pisolithus microcarpus (UFSC-Pt116) in land subject to the sandy process in southern Brazil

Eucalypts is one of the main species used for commercial reforestation in the state of Rio Grande do Sul, Brazil. This study aimed to evaluate the survival and early growth of eucalyptus trees in an area subject to sandy process after three years of growth. The Eucalyptus grandis seedlings were grown in a greenhouse, innoculated or not with the isolated ectomycorrhizal Pisolithus microcarpus (UFSC-Pt116), produced in peat or Entisol. After 120 days, the seedlings were transplanted to an area subject to the sandy process, in the city of São Francisco de Assis, RS. The plants have been evaluated regarding survival, height, stem diameter, nitrogen, phosphorus and potassium levels and total phosphorus, inorganic phosphorus, organic phosphorus and wood production on different days after planting. The seedlings grown on the Entisol which was inoculated with the isolated UFSC-Pt116 presented higher survival rates, height, stem diameter, nitrogen concentration and wood production compared to the non-inoculated seedlings. Inoculation with ectomycorrhizal fungi enhanced the production of E. grandis seedlings in survival rates, height, stem diameter.

Eucalyptus grandis Hill ex Maiden inoculated with Pisolithus microcarpus (UFSC-Pt116) in land subject to the sandy process in southern Brazil

Eucalypts is one of the main species used for commercial reforestation in the state of Rio Grande do Sul, Brazil. This study aimed to evaluate the survival and early growth of eucalyptus trees in an area subject to sandy process after three years of growth. The Eucalyptus grandis seedlings were grown in a greenhouse, innoculated or not with the isolated ectomycorrhizal Pisolithus microcarpus (UFSC-Pt116), produced in peat or Entisol. After 120 days, the seedlings were transplanted to an area subject to the sandy process, in the city of São Francisco de Assis, RS. The plants have been evaluated regarding survival, height, stem diameter, nitrogen, phosphorus and potassium levels and total phosphorus, inorganic phosphorus, organic phosphorus and wood production on different days after planting. The seedlings grown on the Entisol which was inoculated with the isolated UFSC-Pt116 presented higher survival rates, height, stem diameter, nitrogen concentration and wood production compared to the non-inoculated seedlings. Inoculation with ectomycorrhizal fungi enhanced the production of E. grandis seedlings in survival rates, height, stem diameter.

Eucalyptus grandis Hill ex Maiden inoculated with Pisolithus microcarpus (UFSC-Pt116) in land subject to the sandy process in Southern Brazil.

Eucalypts is one of the main species used for commercial reforestation in the Rio Grande do Sul State, Brazil. This study aimed to evaluate the survival and early growth of eucalyptus trees in an area subject to sandy process after three years of growth. The Eucalyptus grandis seedlings were grown in a greenhouse, inoculated or not with the isolated ectomycorrhizal Pisolithus microcarpus (UFSC-Pt116), produced in peat or Entisol. After 120 days, the seedlings were transplanted to an area subject to the sandy process, in the São Francisco de Assis city, RS. The plants have been evaluated regarding survival, height, stem diameter, nitrogen, phosphorus and potassium levels and total phosphorus, inorganic phosphorus, organic phosphorus and wood production on different days after planting. The seedlings grown on the Entisol which was inoculated with the isolated UFSC-Pt116 presented higher survival rates, height, stem diameter, nitrogen concentration and wood production then non-inoculated seedlings. Inoculation with ectomycorrhizal fungi enhanced the production of E. grandis seedlings in survival rates, height, stem diameter.

PtSRR1, a putative Pisolithus tinctorius symbiosis related receptor gene is expressed during the first hours of mycorrhizal interaction with Castanea sativa roots / PtSRR1, um possível receptor simbiose-regulado de Pisolithus tinctorius é expresso nas primeiras horas de interação ectomicorrízica com raízes de Castanea sativa

PtSRR1 EST was previously identified in the first hours of Pisolithus tinctorius and Castanea sativa interaction. QRT-PCR confirmed PtSRR1 early expression and in silico preliminary translated peptide analysis indicated a strong probability that PtSRR1 be a transmembrane protein. These data stimulate the PtSRR1 gene research during ectomycorrhiza formation.

PtSRR1 foi isolado preliminarmente de P. tinctorius nas primeiras horas da interação com raízes de C. sativa. Análises de QRT-PCR confirmaram sua expressão positiva (12 h) e seu peptídeo putativo indicou forte possibilidade para proteína transmembranar. Estes dados estimulam o estudo do PtSRR1 durante a formação de ectomicorrizas.

PtSRR1, a putative Pisolithus tinctorius symbiosis related receptor gene is expressed during the first hours of mycorrhizal interaction with Castanea sativa roots.

PtSRR1 EST was previously identified in the first hours of Pisolithus tinctorius and Castanea sativa interaction. QRT-PCR confirmed PtSRR1 early expression and in silico preliminary translated peptide analysis indicated a strong probability that PtSRR1 be a transmembrane protein. These data stimulate the PtSRR1 gene research during ectomycorrhiza formation.

PtSRR1, a putative Pisolithus tinctorius symbiosis related receptor gene is expressed during the first hours of mycorrhizal interaction with Castanea sativa roots

PtSRR1 EST was previously identified in the first hours of Pisolithus tinctorius and Castanea sativa interaction. QRT-PCR confirmed PtSRR1 early expression and in silico preliminary translated peptide analysis indicated a strong probability that PtSRR1 be a transmembrane protein. These data stimulate the PtSRR1 gene research during ectomycorrhiza formation.

PtSRR1 foi isolado preliminarmente de P. tinctorius nas primeiras horas da interação com raízes de C. sativa. Análises de QRT-PCR confirmaram sua expressão positiva (12 h) e seu peptídeo putativo indicou forte possibilidade para proteína transmembranar. Estes dados estimulam o estudo do PtSRR1 durante a formação de ectomicorrizas.

T-DNA transfer from Agrobacterium tumefaciens to the ectomycorrhizal fungus Pisolithus microcarpus / Transferencia de T-DNA de Agrobacterium tumefaciens al hongo ectomicorrícico Pisolithus microcarpus

The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.

El hongo ectomicorrícico modelo Pisolithus microcarpus aislamiento 441 fue transformado utilizando Agrobacterium tumefaciens LBA 1100 y AGL-1. El marcador de selección fue el gen Shble de Streptoallotecius hidustanus, el cual confiere resistencia a fleomicina, bajo el control del promotor y terminador del gen gpd de Schizophyllum commune. La transformación resultó en clones resistentes a fleomicina comprobándose por PCR la presencia del transgen. La transferencia génica mediada por Agrobacterium podría permitir el desarrollo de la tecnología de interferencia por ARN en P. microcarpus.