Ni2+ toxicity was evaluated in Triticum aestivum L. by its effects on root and shoot length, dry matter production and water content. Over a threshold value of 20 mmol m−3 Ni2+ the degree of toxicity increases as a function of the Ni2+ concentration in the medium. Ni2+‐treated roots show enhanced lipid peroxidation; the higher Ni2+ treatment (40mmol m−3) also increases leakage of K+. In roots and shoots, Ni2+ enhances both guaiacol and syringaldazine extracellular peroxidase activity. The increase in extracellular peroxidase activity is also associated with an increase in the phenolic contents of roots and shoots. The observed growth inhibition might be partly the result of the effect of Ni2+ on cell turgor and cell‐wall extensibility. Intracellular soluble peroxidases are also stimulated by Ni2+; such effects, independently of the substrate, were detected in extracts of Ni2+‐treated shoots at a lower Ni2+ concentration than in the roots. Intracellular peroxidases might act as scavengers of peroxide radicals produced as a result of nickel toxicity.

We studied the involvement of the phytotoxic hydrophobin cerato-ulmin (CU) in pathogenesis and virulence of Dutch elm disease (DED) by expressing its encoding gene (cu) in Ophiostoma quercus, a nonpathogenic species on elm closely related to the DED pathogens O. ulmi and O. novo-ulmi. The production of the toxin was quantitatively determined in culture filtrates and in mycelial extracts of the transformants. Production of CU in vitro was associated with the ability to cause typical DED symptoms, consisting of foliar yellow and wilting and vascular tissue discoloration on a moderately resistant elm genotype. The presence of CU was monitored by enzyme-linked immunosorbent assay in symptomatic leaves of plants inoculated with O. quercus transformants expressing CU and found to be associated with wilt symptoms. In general, the virulence of the cu-expressing transformants, as measured in terms of vascular discoloration and percentage of defoliation, was lower than that of the mildly pathogenic isolate E2 of O. ulmi. However, one transformant (C39) displayed a virulence level intermediate between that of E2 and 182, a highly virulent isolate of O. novo-ulmi. Our results indicate that CU production influences virulence in nonaggressive strains of Ophiostoma fungi.

Natural variants of cerato-platanin (CP), a pathogen associated molecular pattern (PAMP) protein produced by Ceratocystis platani (the causal agent of the plane canker stain), have been found to be produced by other four species of the genus Ceratocystis, including five clones of Ceratocystis fimbriata isolated from different hosts. All these fungal strains were known to be pathogenic to plants with considerable importance in agriculture, forestry, and as ornamental plants. The putative premature proteins were deduced on the basis of the nucleotide sequence of genes orthologous to the cp gene of C. platani; the deduced premature proteins of Ceratocystis populicola and Ceratocystis variospora reduced the total identity of all the others from 87.3% to 60.3%. Cerato-populin (Pop1), the CP-orthologous protein produced by C. populicola, was purified and characterized. Pop1 was a well-structured alpha/beta protein with a different percentage of the alpha-helix than CP, and it self-assembled in vitro in ordered aggregates. Moreover, Pop1 behaved as PAMP, since it stimulated poplar leaf tissues to activate defence responses able to reduce consistently the C. populicola growth.

Cerato-platanin (CP) is a protein produced by Ceratocystis platani, the causal agent of canker stain disease of plane trees. CP is the first member of the 'cerato-platanin family', and its role as a pathogen-associated molecular pattern (PAMP), inducing defence responses both in host and nonhost plants, is established. However, the primary role of CP and its homologues in the fungal life remains unknown. In the present work, we investigated the regulation of the cp gene during the in vitro growth of C. platani in different conditions and under the effect of potential stress factors. Fungal growth and conidiogenesis were also analysed. Results showed that cp is a single-copy gene whose expression level is strictly associated with hyphal growth and with chlamydospores formation. The analysis of a 1368 bp 5'-flanking region revealed putative motifs that could be involved in the regulation of gene expression in response to stress and developmental cues. Taking into account the localization of CP in the fungal cell wall and the recently published 3D structure of the protein, our results support a role for CP in growth and developmental processes of C. platani.

Based on sequence homology, several fungal Cys-rich secreted proteins have been grouped in the cerato-platanin (CP) family, which comprises at least 40 proteins involved mainly in eliciting defense-related responses. The core member of this family is cerato-platanin, a moderately hydrophobic protein with a double ψ-β barrel fold. CP and the recently identified orthologous cerato-populin (Pop1) are involved in host-fungus interaction, and can be considered non-catalytic fungal PAMPs. CP is more active in inducing defense when in an aggregated conformation than in its native form, but little is known about other CP-orthologous proteins. Here, we cloned, expressed, and purified recombinant Pop1, which was used to characterize the protein aggregates. Our results suggest that the unfolded, self-assembled Pop1 is more active in inducing defense, and that the unfolding process can be induced by interaction with hydrophobic inanimate surfaces such as Teflon, treated mica, and gold sheets. In vivo, we found that both CP and Pop1 interact with the hydrophobic cuticle of leaves. Therefore, we propose that the interaction of these proteins with host cuticle waxes could induce unfolding and consequently trigger their PAMP-like activity.

Phyllosticta ampelicida causes black rot disease of Vitis spp. Genetic homogeneity of pathogen populations was investigated by analyzing the number of haplotypes present in infected samples from Europe and America. The fungus was identified from an analysis of the internal transcribed spacer (ITS)1-ITS2 region, and partial sequences of β-tubulin and calmodulin genes. The analysis of nuclear microsatellites applied to strains from Vitis spp. confirmed the existence of a high degree of genetic variability in the fungal populations, revealed four subpopulations, and showed that strains from America are distinct from the European ones. Furthermore, the results obtained by landscape genetics showed that there were different introductions of the pathogen in the main vine areas of Europe, confirming what was observed in the first reports of the disease. The genetic variability of the fungus revealed by this study confirms the ability to generate new haplotypes by sexual reproduction. The difference found between the European populations and the American one confirms that the pathogen originated from America.

Cerato-platanin (CP), a protein previously isolated from the ascomycete Ceratocystis fimbriata f.sp. pla-tani (Cfp), the causal agent of plane canker stain, has been studied with regard its phytotoxic effect in plane leaf cells by transmission electron microscopy. When infiltrated in plane leaf mesophyllum, pure cerato-platanin induced, 24 and 48 h after treatment, several negative responses in palisade and spongy tissues. One of the most important responses consisted, already after 24 h, in a great production of primary starch in these tissues. Moreover, the ultrastructural observations evidenced numerous other symptoms, such as strong alterations in chloroplasts, nuclei and in the cytoplasm. These changes were particularly serious in spongy parenchyma cells. In addition, these cells appeared strongly squashed with a reduced and completely disorganized protoplast.

a b s t r a c tPrevious work had shown that a sequence homologous to the gene encoding class II hydrophobin cerato-ulmin from the fungus Ophiostoma novo-ulmi, the causal agent of Dutch Elm Disease (DED), was present in a strain of the unrelated species Geosmithia species 5 (Ascomycota: Hypocreales) isolated from Ulmus minor affected by DED. As both fungi occupy the same habitat, even if different ecological niches, the occurrence of horizontal gene transfer was proposed. In the present work we have analysed for the presence of the cerato-ulmin gene 70 Geosmithia strains representing 29 species, isolated from different host plants and geographic locations. The gene was found in 52.1 % of the strains derived from elm trees, while none of those isolated from nonelms possessed it. The expression of the gene in Geosmithia was also assessed by real time PCR in different growth conditions (liquid culture, solid culture, elm sawdust, dual culture with O. novo-ulmi), and was found to be extremely low in all conditions tested. On the basis of these results we propose that the cerato-ulmin gene is not functional in Geosmithia, but can be considered instead a marker of more extensive transfers of genetic material as shown in other fungi.