SURROGATE MOTHER FOR ENDAGERED Cupressus dupreziana  (2002)

In the cypress improvement programme for resistance against canker disease, caused by Seiridium cardinale, controlled crosses among cypress species were done by the IPP. The hybrids Cupressus sempervirens (as female) x C. dupreziana (as male) aroused great interest, becuase some morphological traits of hybrids were always identical to those of the paternal tree, C. dupreziana. A team, constituted by Italian and French biologists, geneticists and sylviculturists, examined six 15-year-old families obtained by controlled crosses of 6 different clones of C. sempervirens (as female) and C. dupreziana (as male). C. dupreziana is native to the Tassili N’Ajjer (figure 1) desert of Algeria and is one of the most threatened trees in the world. The following morphological characteristics distintive of the parental species were measured for the hybrids and the parents:
- orientation of terminal twigs : in one plane in C. dupreziana, in all directions in C. sempervirens;
- cone size: always larger in C. sempervirens;
- percentage of filled seed: very low in C. dupreziana;
- endosperm ploidy levels: only even levels:2n, 4n, 6n…in C. dupreziana;
- pollen diameter: 38 ìm in C. dupreziana and 28 ìm in C. sempervirens;
- pollen ploidy level: diploid in C. dupreziana.
For all these characters all the hybrids resulted identical to the male tree, C. dupreziana. Genetic diversity was assessed by using two markers: isozymes in only one hybrid family ( seven polymorphic sistems: Fest, Idh,Lap,6Pgd, Pgi, Pgm and Skdh) and RAPD, four operon primers:OPA-08, OPA-15, OPA-18 and OPR-07) in four hybrid families.
A biparental codominant inheritance was previously reported in C. sempervirens for these isozymes, whereas the genetic control of the RAPD markers was unknown. The markers allowed identification of all the parents. Progeny had a single genetic pattern that was strictly identical to that of the father, C. dupreziana. These results confirm our hypothesis that pollen development in C. dupreziana is apomictic. This leads to the production of embryos that are genetically unrelated to the other seed components (maternal sporophyte and gametophyte). The results explain very well the previosly discovered significant anomalies in the reproductive structure of C. dupreziana. In this variant, viable pollen (male gametophyte) is diploid, embryos do not have the same allozymes as their mothers, and the endosperm (seed nutritive tissue) is not haploid, although in Gymnosperms it is derived solely from the female gametophyte. In our tests another cypress species, C. sempervirens, resulted a surrogate mother for this embryogenic pollen. This is, to our knowledge, the first report of paternal apomixis in plants. Inbreeding in small size popolation of C. dupreziana (231 individuals) reduces fitness and suvival in the progeny and increases the risk of extintion. Paternal apomixis can be considered as a vegetative multiplication, which can overpass the inbreeding risks. Now our hypothesis is that this deviant reproductive pattern evolved in response to the reduction of the C. dupreziana population size.
See for details “Nature, where the work was published (5 July 2001)

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Fungi as potential agents of bioremediation of asbestos-contaminated soil  (2002)

Industrialized nations' passing flirtation with asbestos has left a nasty legacy. Exposure to airborne asbestos fibrils causes in fact asbestosis and malignancies such as bronchogenic carcinoma and pleural mesothelioma. Although extraction and use of asbestos have been banned in many countries, substantial amounts of fibres have remained on sites previously occupied by dismissed asbestos industries. Cleaning up contaminated soil around defunct asbestos mines and factories is a lot harder than removing asbestos sheets from buildings, and the widely-dispersed fibres can easily become airborne if disturbed. The toxic effects of inhaled asbestos fibres rely on both physical and chemical factors. The surface properties of asbestos fibres, and in particular iron, play a role in pathogenicity ( ). Crocidolite, one of the most potently carcinogenic forms of asbestos, contains up to 29 % iron, that originates highly reactive centres when occurring at the surface in poorly co-ordination state. This can cause the formation of highly reactive free radicals that damage DNA and eventually trigger cancers. Experiments in vitro demonstrate that iron removal makes the asbestos considerably less hazardous by reducing their potential to generate radicals and to damage DNA ( ). Most soil microorganisms need iron for their own metabolism, so some have very effective ways of scavenging it from their environment. Plants, bacteria and fungi can release potent chelators such as siderophores and polycarboxylic acids, that grab hold of iron atoms in soil minerals and bring it into soluble forms.
We have focused on fungi because these organisms display interesting features for bioremediation: they are found in all ecosystems and, through the formation of extensive hyphal networks, they explore large soil volumes. In addition, their biodiversity provides a huge reservoir of genes and functions. We thus investigated growth of some soil fungi in the presence of crocidolite fibres and their ability to release iron from this material. We demonstrated that fungi can steal considerable amounts of iron from crocidolite (Figure 1). The best iron-gatherers among the species tested were Fusarium oxysporum, Mortierella hyalina and Oidiodendron maius, a mycorrhizal fungus. Some species were still sucking up iron after more than seven weeks. In addition, fungal hyphae can form a web of thin strands that bind asbestos fibres, making them less liable to escape into the air (Figure 1, inset). The action of fungal chelators contribute s to modify the fibre surface in vitro, depriving it of the active sites likely involved in the triggering of the carcinogenic mechanisms. After exposure to fungi, in fact, the iron-stripped fibres could not generate reactive free radicals ( ). We suggest, as also reported on Nature Science Update (http://www.nature.com/nsu/ 030120/030120-2.html), that if iron could be progressively extracted from the fibres dispersed in the environment, by native or introduced fungi, consequent changes in the nature of the fibre surface could likely result in a decrement in their carcinogenic potential.

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THE HIGHEST GENETIC FOREST DIVERSITY IS FOUND IN THE CENTRAL EUROPEAN FORESTS   (2003)

Cyclic environmental changes have occurred during the last two million years. Living organisms react to changing phases through adaptation strategies, otherwise they get extinct. Forest trees are among the most ancient living organisms and in many cases they symbolise the last representatives of taxonomic units that underwent severe selection and/or extinction. The present distribution of trees in the forest landscape is the result of population dynamics experienced during the last millennia. In order to understand the mechanisms determining the evolution and the diversity of forest tree populations, it is important to consider historical factors, such as climatic and geological events, and evidences provided by fossil pollen records. The availability of fossil pollen records is restricted to few taxa, i.e., those producing large amount of pollen and characterised by wind pollination. Recently, the application of molecular markers to population genetics has provided additional research tools.
Important phylogeographic studies have been made on plant species with the analysis of organellar (chloroplast and mitochondria) DNA. Chloroplast and mitochondria represent haploid genomes, uniparentally transmitted through the generation: mitochondria are passed though the female parent in both animal and plant organisms, chloroplast are generally maternally inherited in angiosperms (through seed), and parentally inherited in gymnosperms (though pollen). For maternally inherited genomes, seed dispersal mechanisms determine the geographic structure of genetic diversity. Furthermore, the influence of human activities plays an important role in shaping the genetic diversity of forest tree populations.
Within the frame of the European project CYTOFOR (Measuring molecular differentiation of European deciduous forests for conservation and management, FAIR5 - CT97 – 3795), we analysed chloroplast diversity in twenty-two angiosperm shrub and tree species. These were sampled in the same 25 European forests selected on the basis of their high species richness and limited human influence. The innovative aspect of this study is the multispecies approach: genetic diversity was described not only for single species, but also at the forest level. To compare forests with each other, we calculated the mean number of haplotypes and within-population gene diversity by averaging across species in each forest. Furthermore, a measure that expresses the average genetic divergence of the forest from all remaining populations was also calculated.
Seed dispersal mechanisms influence the distribution of the haplotipic diversity. Low values of genetic differentiation (expressed by the coefficient GST), indicating high levels of gene flow through seeds, were found in Salix and in Populus, genera characterized by light, wind-dispersed seeds The species characterised by animal-ingested seeds also tended to have below-average values. In contrast, species with animal-cached seeds (i.e., nuts) exhibited higher than average values.
The highest values of genetic divergence were observed in Corsica, Italy and the Balkans, including Croatia and Romania. This finding is in agreement with the location of the most important glacial refugia. On the other hand, lower values of genetic divergence were estimated in the other European regions (Fig. 1). Patterns of diversity across forests were very different; both mean number of haplotypes (Fig. 2) and gene diversity were higher in Central F

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Arbuscular mycorrhizal (AM) fungi: Snapshots 2004  (2003)

Arbuscular mycorrhizal fungi (AM) are indeed mysterious and still surprising organisms. Plenty of papers have recently provided new insights on these organisms. The classification of AM fungi has been reviewed even if it's still under debate. The phosphate transporters in AM fungi and their impact on the phosphate transfer in plant is giving new exciting glances to a crucial step of plant physiology. Other fundamental aspects, such as lipids and carbohydrates metabolism and C translocation, have been clarified by means of NMR experiments. However, the mother of all the questions is still unsolved: what about the genome in AM fungi? There are two levels hidden behind this same question: i) the organization and the complexity of the fungal genome (genome size, ploidy, repeated sequences and so on) and ii) the affordability of a genome sequencing project like those already available for Saccharomyces cerevisiae or Neurospora crassa. The first level is a priority to understand the accomplishment of the AM life cycle, the genetic polymorphisms, as well as the variability which has already been reported at least in ribosomal genes. All these data have so far supported the concept that heterokaryosis, the coexistence of different nuclei in cells, occurs troughout the AM fungal life. Very recent results opened the way to the alternative hypothesis of homokaryosis. The second level will be crucial to compare AM fungi with saprotrophic or pathogenic fungi and to understand how they impact on plant development. Recently it was demonstrated that Glomus intraradices is haploid and has a small genome size in the lower limit for eukaryotes. It will be very important to see whether low values are confirmed also for other AM species: this will allow us to understand whether there is a relationship between biotrophism and genome size. The genomics and functional genomics of mycorrhizal plants are currently important tools to investigate the influence of the fungus in comparison to non-mycorrhizal plants. It has been successfully demonstrated that EST-based approaches are effective in monitoring the interaction between symbionts, but information is mostly about the plant or the mycorrhiza taken as a whole, rather than about the fungus itself. Fungal transcripts coming from mycorrhizal roots are evaluated as not more than the 5%. Therefore, a more direct way is to study the fungus alone, by sequencing the genome and collecting ESTs. In the last months the proposal to sequencing G. intraradices genome has been approved, and this project will be carried out during 2004. In addition to the extraordinary possibility of having a global genetic blueprint for an AM fungus, we can expect to have some answers to one of the basic questions we have: is there a genetic basis for biotrophy? By comparing the genome of G. intraradices with that of a saprobic fungus, we will be able to see whether some genes, crucial for an independent life, are missing and whether the host plant complements the missing functions.

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