Stefania Tegli

Fluctuation of bacteria isolated from elm tissues during different seasons and from different plant organs

In this work we isolated a culturable endophytic aerobic heterotrophic bacterial community from the stem and root tissues of elm trees (Ulmus spp.) and analyzed its fluctuations. A total of 724 bacterial isolates were collected at different times (April, June, September and December) from two elm trees, one infected with Elm Yellows phytoplasmas, and one which was healthy-looking. The isolates were grouped into 82 haplotypes, identified by means of amplified ribosomal DNA restriction analysis (ARDRA) using the restriction enzyme AluI, suggesting that the genetic diversity of the bacterial community was very high. The taxonomic position of the isolates belonging to the twelve main haplotypes, representing more than 72% of the total population, was determined by 16S rDNA sequencing. The main genera were Bacillus, Curtobacterium, Pseudomonas, Stenotrophomonas, Sphingomonas, Enterobacter, and Staphylococcus. The fluctuations in the bacterial community, determined by different parameters (seasonal changes, plant organ, presence of phytoplasmas) were studied, revealing that they were influenced both by variations in temperature (warm or cold according to the season) and by the organ examined (roots or stems). The role of the phytopathogenic status in these fluctuations was also discussed.

Sequence analysis of ITS ribosomal DNA in five Phaeoacremonium species and development of a PCR-based assay for the detection of P. chlamydosporum and P. aleophilum in grapevine tissue

Phaeoacremonium aleophilum and P. chlamydosporum are two recently described mitosporic fungi that are involved in the development of symptoms of esca disease and of a decline of young grapevines previously named “black goo”. The Internal Transcribed Spacers (ITS) 1 and 2, plus the interveining 5.8S gene, of ribosomal DNA (rDNA) of representative isolates of the two species and, for comparison, of isolates of the congeneric species P. angustius, P. inflatipes and P. rubrigenum were amplified by Polymerase Chain Reaction (PCR) using the ITS4 and ITS5 universal primers. The size of the entire ITS region (ITS1-5.8S-ITS2), plus the 3’ end of 18S rDNA and the 5’ end of 28S rDNA, was estimated to be about 620 bp, on gel electrophoresis, for all the Phaeacremonium species tested. Eleven restriction enzymes were used singularly in the digestion of the ITS region of 30 isolates of P. chlamydosporum, 16 of P. aleophilum, 2 of P. angustius, 2 of P. inflatipes and 1 of P. rubrigenum. No length-polymorphism could be detected within species (except for P. aleophilum), but there were quite strong differences between species. The PCR products of ITS region of ten representative isolates for the five Phaeoacremonium species were sequenced, and the sequences aligned and compared. Two main groups were clearly distinguishable, one formed by P. chlamydosporum, and the other by P. aleophilum, P. angustius, P. inflatipes and P. rubrigenum, with an homology between the two groups ranging from 64.5% to 66.5%. The sequences of ITS region were used to design two pairs of primers, Pal1N/ Pal2 and Pch1/Pch2, each of which was subsequently shown to be specific for the amplification of predicted-size fragments from genomic DNA of P. aleophilum and P. chlamydosporum, respectively. The identity of the amplified fragments was confirmed by sequencing. The primer pairs were further tested using as template DNA extracted from healthy grapevines and from other fungi commonly isolated from esca-diseased grapevine plants but no amplification was observed. The PCR protocol was shown to be quite sensitive (10 pg of DNA) and able to specifically detect P. chlamydosporum and P. aleophilum in artificially inoculated grapevine plants.

Naturally occurring non cerato-ulmin producing mutants of Ophiostoma novo-ulmi are pathogenic but lack aerial mycelium

Cerato-ulmin is a protein implicated as a major toxin in the development of Dutch elm disease symptoms in elms infected with Ophiostoma novo-ulmi. O. novo-ulmi isolates typically produce fibrous-striate aerial mycelium on malt extract agar and secrete high levels of cerato-ulmin in liquid medium. However, two different genotypes of O. novo-ulmi (NAN race) from a population sample in Portugal, isolates MAFf8 and PG470, exhibited unusual flat-waxy ‘non-aerial mycelial’ colony types and were found to produce no detectable cerato-ulmin. The two isolates otherwise behaved as normal O. novo-ulmi isolates, including being highly pathogenic to Ulmus procera . When MAFf8 and PG470 were crossed with wild-type NAN O. novo-ulmi isolates, non-aerial mycelial colony phenotype and non cerato-ulmin production was shown in each case to be controlled by a single pleiotropic mutation, termed cu − . A further cross showed the cu − locus was probably allelic in the two isolates. The influence of the cu − locus on colony phenotypes in O. novo-ulmi supports the view that cerato-ulmin is a fungal hydrophobin. The normal pathogenic ability of the two cu − isolates raises questions about the proposed role of cerato-ulmin as a wilt toxin.

Cerato-platanin, the first member of a new fungal protein family: cloning, expression, and characterization.

The ascomcete Ceratocystis fimbriata, the causal agent of “canker stain disease,” secretes a protein of 12.4 kDa that elicits phytoalexin synthesis and plant cell death. This protein, named cerato-platanin (CP), is also located in the cell walls of ascospores, hyphae, and conidia; it contains four cysteines (S-S bridged) and is moderately hydrophobic. The cp gene consists of a single exon and has 42 bp codifying for a signal peptide of 14 residues. The recombinant protein was obtained by cloning the cp gene of the mature protein in Escherichia coli (BL21), and a refolding step was needed to achieve the native active form. In the European Molecular Biology data bank, CP is reported as the first member of the CP family; this is the first example of an set of secreted fungal proteins whose primary structure is very similar. Nonetheless, the data also revealed some structural and functional features that make CP simlar to proteins of the hydrophobin family.

High-Resolution Melting Analysis as a Powerful Tool to Discriminate and Genotype Pseudomonas savastanoi Pathovars and Strains

Pseudomonas savastanoi is a serious pathogen of Olive, Oleander, Ash, and several other Oleaceae. Its epiphytic or endophytic presence in asymptomatic plants is crucial for the spread of Olive and Oleander knot disease, as already ascertained for P. savastanoi pv. savastanoi (Psv) on Olive and for pv. nerii (Psn) on Oleander, while no information is available for pv. fraxini (Psf) on Ash. Nothing is known yet about the distribution on the different host plants and the real host range of these pathovars in nature, although cross-infections were observed following artificial inoculations. A multiplex Real-Time PCR assay was recently developed to simultaneously and quantitatively discriminate in vitro and in planta these P. savastanoi pathovars, for routine culture confirmation and for epidemiological and diagnostical studies. Here an innovative High-Resolution Melting Analysis (HRMA)-based assay was set up to unequivocally discriminate Psv, Psn and Psf, according to several single nucleotide polymorphisms found in their Type Three Secretion System clusters. The genetic distances among 56 P. savastanoi strains belonging to these pathovars were also evaluated, confirming and refining data previously obtained by fAFLP. To our knowledge, this is the first time that HRMA is applied to a bacterial plant pathogen, and one of the few multiplex HRMA-based assays developed so far. This protocol provides a rapid, sensitive, specific tool to differentiate and detect Psv, Psn and Psf strains, also in vivo and against other related bacteria, with lower costs than conventional multiplex Real-Time PCR. Its application is particularly suitable for sanitary certification programs for P. savastanoi, aimed at avoiding the spreading of this phytopathogen through asymptomatic plants.

Phenols and Stilbene Polyphenols in the Wood of Esca-Diseased Grapevines

Trans-resveratrol and e-viniferin, already described as stress metabolites produced by the leaves of Vitis vinifera in response to fungal infection, UV irradiation, and incubation by chemicals, have been detected in the wood of healthy as well as in the brown-red wood of esca-diseased grapevines. Brown-red wood is considered an initial symptom of esca in grapevine. Resveratrol was the predominant component although e-viniferin was also accumulated in appreciable quantities. The biological significance of the production of resveratrol and e-viniferin is discussed.

Fluctuation of endophytic bacteria and phytoplasmosis in elm trees

A total of 658 heterotrophic bacterial colonies isolated from phloem tissues of roots and branches in four months (April, June, September and December) from two elm plants, one of which affected by phytoplasmosis, were typed by means of ARDRA. This analysis revealed the existence of a high degree of variability within the community and was able to detect 84 different ARDRA groups. The Analysis of Molecular Variance was applied to ARDRA patterns to analyze the differentiation between communities isolated from the various samplings. Data obtained were compared with those from a previous work (Mocali et al. 2003). Results indicated that plants with symptoms of phytoplasmosis showed marked alterations in the extent of the fluctuations of the community along the seasons in the different plant organs.

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.

Isolation and characterization of non cerato-ulmin producing laboratory induced mutants of Ophiostoma novo-ulmi

Five laboratory mutants of Ophiostoma novo-ulmi EAN isolate H328 were obtained from uv-irradiated and unirradiated blastoconidia. Although the mutants were initially selected on the basis of various characteristics, including failure to produce cerato-ulmin (CU) in the culture filtrate, growth-temperature responses and colony morphology, they all were eventually also found unable to produce CU when grown in liquid shake culture at 23 °C and 33°, or they produced CU only in negligible amounts. This association suggests possible linkages between CU production and other fungal metabolic pathways. Genetic analysis of crosses between the mutants and a wild type isolate of EAN O. novo-ulmi suggested that each mutant involved a change at a single-locus, at least as far as segregations for colony morphology were concerned. Pathogenicity trials using the five mutants were carried out on four-year old Ulmus carpinifola and U. procera . Two out of the five mutants showed a significant reduction in pathogenicity in comparison to the O. novo-ulmi wild type isolate H328.

Comparative determination of cerato-ulmin on cell surface and in mycelial extracts of pathogenic and non-pathogenic Ophiostoma species

Cerato-ulmin (CU) presence was monitored on cell surface and quantitatively determined in mycelial extracts of the elm pathogens Ophiostoma novo-ulmi (races EAN and NAN) and O. ulmi and of the non-pathogenic O. piceae . CU was detected on the surfaces of Ophiostoma novo-ulmi (races EAN and NAN) and, for the first time, of the weak Dutch elm disease pathogen O. ulmi and the non-pathogen O. piceae . Quantitative determination of CU content in the mycelial extracts of the three species showed that high CU cellular content is associated with high CU content in culture filtrates. The content of CU in biomasses and in culture filtrates was influenced by temperature, growth phase and fungal species or race. CU synthesis occurred during the stationary phase and in the late logarithmic phase when fungi were grown at 23° and 32°C, respectively. High temperatures of growth (32°) did not have a negative effect on the cellular CU content but severely hampered CU secretion in high CU-producers O. novo-ulmi isolates.