Cecilia Comparini

Functional Expression of the Gene cu, Encoding the Phytotoxic Hydrophobin Cerato-ulmin, Enables Ophiostoma quercus, a Nonpathogen on Elm, to Cause Symptoms of Dutch Elm Disease

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.

New proteins orthologous to cerato-platanin in various Ceratocystis species and the purification and characterization of cerato-populin from Ceratocystis populicola

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 α/β protein with a different percentage of the α-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.

Effect of temperature on growth and cerato-ulmin production of Ophiostoma novo-ulmi and O. ulmi

Ophiostoma novo-ulmi and O. ulmi , previously named ‘aggressive’ and ‘non-aggressive’ subgroups of the old species O. ulmi , are distinguished by a wide range of morphological, molecular, genetical and physiological characters, observed both in vivo and in vitro . They show different virulence on elms of moderate resistance, that seems to be correlated with the ability to synthesize phytotoxic compounds, of which the most important is cerato-ulmin (CU). To date, O. novo-ulmi isolates have been considered the greatest producers of CU in contrast to O. ulmi isolates, that synthesize very little or no toxin. We demonstrate that the synthesis of CU is temperature-dependent and that the two species seem to have different temperature optima for its production. However, while the difference in CU production is linked to temperature, it is not a consequence of differences in fungus growth in liquid shake culture caused by these temperature differences.

Cerato-Populin and Cerato-Platanin, Two Non-Catalytic Proteins from Phytopathogenic Fungi, Interact with Hydrophobic Inanimate Surfaces and Leaves

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.

Widespread horizontal transfer of the cerato-ulmin gene between Ophiostoma novo-ulmi and Geosmithia species

Previous 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.

Multiple Forms of Pectin-Degrading Enzymes Produced by Intersterile Groups P, S and F of "Heterobasidion annosum" (Fr.) Bref

The time-course of polygalacturonase (PG) and pectin lyase (PNL) activities shown in vitro by several isolates belonging to the intersterile groups (IGs) S and F of Heterobasidion annosum are reported and discussed in relation to their mycelial growth and the changes in the pH and viscosity of the growth medium they produce. IG-S, characterized by a narrow host range, always produced low levels of enzymatic activity. IG-F grew more abundantly and faster than IG-P and IG-S. In the nutrient medium containing pectin as the sole carbon source. PG activity shown by IG-F per unit of mycelial mass was similar to that of IG-P, but different from that of IG-S; by contrast, the specific PNL activity of IG-F was comparable to that of IG-S, but not to that of IG-P. PG, PNL and pectin methylesterase (PME) activities were separated by non-denaturing isoelectric focusing-polyacrylamide gel electrophoresis; to detect the isozymes, agarose overlays containing the appropriate substrate were stained, in a ruthenium red solution. Each IGs behaved differently from the others, and the variations among them were statistically significant. The isozymatic patterns were also very strongly influenced by the culture period. IG-S, unlike the two other IGs, always showed a single PG band with pI 4.7; an attempt to carry out a partial purification of the PG band with pI 4.7 is shown. The results of the present study open the avenue to obtaining more information about the role of pectinolytic enzymes in the root rot caused by H. annosum.

Isolation of the orthologue of the cerato-ulmin gene in Ophiostoma quercus and characterization of the purified protein

Ophiostoma quercus is an ophiostomatoid fungus strictly related to the Ophiostomas (O. ulmi, O. novo-ulmi, and O. himal-ulmi) that cause Dutch elm disease (DED). O. quercus has a number of morphological characteristics in common with the DED pathogens, and is a well-known and economically important sapstaining fungus occurring worldwide on hardwoods and commercially produced pines, and causes typical cankers on oak stems. In elm trees O. quercus can survive for months without causing any disease symptoms. DED fungi produce cerato-ulmin (CU), a class II hydrophobin, which is generally considered as the main toxin potentially involved in various phases of the DED pathogenesis. In the present work we isolated and sequenced the orthologue of the cu gene in the O. quercus isolates H988, H1042, and H2053. Moreover the CU protein from O. quercus isolate H988 was also purified and characterized. Sequence analysis showed that there is a pronounced difference between the whole cu gene region of O. quercus and the homologous fragments of the DED-causing species O. ulmi, O. novo-ulmi, and O. himal-ulmi. It also appeared that differences in the structural conformation of the promoter were unlikely to play a role in the modulation of the transcript level and that, for O. quercus, differences in CU production did not result from the potential different regulation levels. Clear differences were shown in the transcriptional unit of the cu genes and in the amino acid sequences among all the CUs. The purified O. quercus CU was separated using matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) spectrometry into seven forms of increasing molecular weight from 7190 to 7724Da. The hydrophobicity profiles indicated that two regions of the O. quercus CU protein were more hydrophobic than the corresponding regions of the CUs of the DED fungi. The O. quercus CUs had theoretical isoelectric point values similar to those of the DED fungi. Finally, the contradiction between the consistent differences between these four Ophiostoma species in the cu gene region and in the CU proteins and their strict phylogenetic relationship is discussed.

Genetic Variability of Phyllosticta ampelicida, the Agent of Black Rot Disease of Grapevine

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.

Geosmithia-Ophiostoma: a New Fungus-Fungus Association

In Europe as in North America, elms are devastated by Dutch elm disease (DED), caused by the alien ascomycete Ophiostoma novo-ulmi. Pathogen dispersal and transmission are ensured by local species of bark beetles, which established a novel association with the fungus. Elm bark beetles also transport the Geosmithia fungi genus that is found in scolytids’ galleries colonized by O. novo-ulmi. Widespread horizontal gene transfer between O. novo-ulmi and Geosmithia was recently observed. In order to define the relation between these two fungi in the DED pathosystem, O. novo-ulmi and Geosmithia species from elm, including a GFP-tagged strain, were grown in dual culture and mycelial interactions were observed by light and fluorescence microscopy. Growth and sporulation of O. novo-ulmi in the absence or presence of Geosmithia were compared. The impact of Geosmithia on DED severity was tested in vivo by co-inoculating Geosmithia and O. novo-ulmi in elms. A close and stable relation was observed between the two fungi, which may be classified as mycoparasitism by Geosmithia on O. novo-ulmi. These results prove the existence of a new component in the complex of organisms involved in DED, which might be capable of reducing the disease impact.

Correction to: Geosmithia-Ophiostoma: a New Fungus-Fungus Association

The article Geosmithia-Ophiostoma: a New Fungus-Fungus Association, written by Alessia L. Pepori, Priscilla P. Bettini, Cecilia Comparini, Sabrina Sarrocco, Anna Bonini, Arcangela Frascella, Luisa Ghelardini, & Aniello Scala, Giovanni Vannacci, Alberto Santini.