Matteo Cerboneschi

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.

Development of a versatile tool for the simultaneous differential detection of Pseudomonas savastanoi pathovars by End Point and Real-Time PCR

BackgroundPseudomonas savastanoi pv. savastanoi is the causal agent of olive knot disease. The strains isolated from oleander and ash belong to the pathovars nerii and fraxini, respectively. When artificially inoculated, pv. savastanoi causes disease also on ash, and pv. nerii attacks also olive and ash. Surprisingly nothing is known yet about their distribution in nature on these hosts and if spontaneous cross-infections occur. On the other hand sanitary certification programs for olive plants, also including P. savastanoi, were launched in many countries. The aim of this work was to develop several PCR-based tools for the rapid, simultaneous, differential and quantitative detection of these P. savastanoi pathovars, in multiplex and in planta.ResultsSpecific PCR primers and probes for the pathovars savastanoi, nerii and fraxini of P. savastanoi were designed to be used in End Point and Real-Time PCR, both with SYBR® Green or TaqMan® chemistries. The specificity of all these assays was 100%, as assessed by testing forty-four P. savastanoi strains, belonging to the three pathovars and having different geographical origins. For comparison strains from the pathovars phaseolicola and glycinea of P. savastanoi and bacterial epiphytes from P. savastanoi host plants were also assayed, and all of them tested always negative. The analytical detection limits were about 5 - 0.5 pg of pure genomic DNA and about 102 genome equivalents per reaction. Similar analytical thresholds were achieved in Multiplex Real-Time PCR experiments, even on artificially inoculated olive plants.ConclusionsHere for the first time a complex of PCR-based assays were developed for the simultaneous discrimination and detection of P. savastanoi pv. savastanoi, pv. nerii and pv. fraxini. These tests were shown to be highly reliable, pathovar-specific, sensitive, rapid and able to quantify these pathogens, both in multiplex reactions and in vivo. Compared with the other methods already available for P. savastanoi, the identification procedures here reported provide a versatile tool both for epidemiological and ecological studies on these pathovars, and for diagnostic procedures monitoring the asymptomatic presence of P. savastanoi on olive and oleander propagation materials.

Effect of the plant flavonoid luteolin on Ensifer meliloti 3001 phenotypic responses

Background and aimsThe establishment of a successful symbiosis between the nitrogen-fixing bacterium Ensifer meliloti and compatible host legumes (Medicago spp.) depends on a complex molecular signal exchange. The early stage of signaling involves the release from plant roots of the flavonoid luteolin, which in turn induces the expression of rhizobia nodulation (nod) genes required for root infection and nodule development. To date, the bacterial response to the luteolin perception has been characterized in detail as far as gene expression is concerned. Nevertheless, despite this molecular information, a global view on E. meliloti phenotypes affected by the plant signal luteolin is still lacking. Therefore, an extensive phenotypic investigation of luteolin effect on the nitrogen-fixing E. meliloti 3001 has been performed.MethodsA thousand different growth conditions, including sensitivity to osmolites, pH stresses, antibiotics and toxic compounds, were tested by the application of the high-throughput Phenotype MicroArray (PM) technology, as well as by several dedicated assays to evaluate growth stimulation, motility, biofilm formation, N-acyl homoserine lactones and Indole-3- acetic acid (IAA) production.ResultsResults revealed that the plant signal luteolin affected a wide spectrum of E. meliloti 3001 phenotypes. E. meliloti 3001 displayed an enhanced resistance phenotype in the presence of luteolin toward a broad set of chemicals including several antibiotics, toxic ions, respiration inhibitors, membrane damagers, DNA intercalants and other potential antimicrobial agents. Moreover, the presence of luteolin significantly reduced overall AHLs production, as well as the lag phase in relation to the starting cellular density, the motility and biofilm formation under nutrient-limited growth conditions. An effect on E. meliloti indole-3-acetic acid (IAA) production was also detected in vitro as a response to luteolin.ConclusionsOverall, these findings suggest that the plant signal luteolin triggers a broad response in E. meliloti 3001, which was shown to be dependent on nutritional conditions sensed by the bacterium, pointing out a wide role in modifying rhizobial phenotypes, possibly in relation to plant root association and then symbiotic interaction.

Decolorization of acid and basic dyes: understanding the metabolic degradation and cell-induced adsorption/precipitation by Escherichia coli

Escherichia coli strain DH5α was successfully employed in the decolorization of commercial anthraquinone and azo dyes, belonging to the general classes of acid or basic dyes. The bacteria showed an aptitude to survive at different pH values on any dye solution tested, and a rapid decolorization was obtained under aerobic conditions for the whole collection of dyes. A deep investigation about the mode of action of E. coli was carried out to demonstrate that dye decolorization mainly occurred via three different pathways, specifically bacterial induced precipitation, cell wall adsorption, and metabolism, whose weight was correlated with the chemical nature of the dye. In the case of basic azo dyes, an unexpected fast decolorization was observed after just 2-h postinoculation under aerobic conditions, suggesting that metabolism was the main mechanism involved in basic azo dye degradation, as unequivocally demonstrated by mass spectrometric analysis. The reductive cleavage of the azo group by E. coli on basic azo dyes was also further demonstrated by the inhibition of decolorization occurring when glucose was added to the dye solution. Moreover, no residual toxicity was found in the E. coli-treated basic azo dye solutions by performing Daphnia magna acute toxicity assays. The results of the present study demonstrated that E. coli can be simply exploited for its natural metabolic pathways, without applying any recombinant technology. The high versatility and adaptability of this bacterium could encourage its involvement in industrial bioremediation of textile and leather dyeing wastewaters.

Water recycle as a must: decolorization of textile wastewaters by plant-associated fungi

Textile dye effluents are among the most problematic pollutants because of their toxicity on several organisms and ecosystems. Low cost and ecocompatible bioremediation processes offer a promising alternative to the conventional and aspecific physico‐chemical procedures adopted so far. Here, microorganisms resident on three real textile dyeing effluent were isolated, characterized, and tested for their decolorizing performances. Although able to survive on these real textile‐dyeing wastewaters, they always showed a very low decolorizing activity. On the contrary, several plant‐associated fungi (Bjerkandera adusta, Funalia trogii, Irpex lacteus, Pleurotus ostreatus, Trametes hirsuta, Trichoderma viride, and Aspergillus nidulans) were also assayed and demonstrated to be able both to survive and to decolorize to various extents the three effluents, used as such in liquid cultures. The decolorizing potential of these fungi was demonstrated to be influenced by nutrient availability and pH. Best performances were constantly obtained using B. adusta and A. nidulans, relying on two strongly different mechanisms for their decolorizing activities: degradation for B. adusta and biosorption for A. nidulans. Acute toxicity tests using Daphnia magna showed a substantial reduction in toxicity of the three textile dyeing effluents when treated with B. adusta and A. nidulans, as suggested by mass spectrometric analysis as well.

Global Analysis of Type Three Secretion System and Quorum Sensing Inhibition of Pseudomonas savastanoi by Polyphenols Extracts from Vegetable Residues

Protection of plants against bacterial diseases still mainly relies on the use of chemical pesticides, which in Europe correspond essentially to copper-based compounds. However, recently plant diseases control is oriented towards a rational use of molecules and extracts, generally with natural origin, with lower intrinsic toxicity and a reduced negative environmental impact. In this work, polyphenolic extracts from vegetable no food/feed residues of typical Mediterranean crops, as Olea europaea, Cynara scolymus, and Vitis vinifera were obtained and their inhibitory activity on the Type Three Secretion System (TTSS) and the Quorum Sensing (QS) of the Gram-negative phytopathogenic bacterium Pseudomonas savastanoi pv. nerii strain Psn23 was assessed. Extract from green tea (Camellia sinensis) was used as a positive control. Collectively, the data obtained through gfp-promoter fusion system and real-time PCR show that all the polyphenolic extracts here studied have a high inhibitory activity on both the TTSS and QS of Psn23, without any depressing effect on bacterial viability. Extracts from green tea and grape seeds were shown to be the most active. Such activity was confirmed in planta by a strong reduction in the ability of Psn23 to develop hyperplastic galls on explants from adult oleander plants, as well as to elicit hypersensitive response on tobacco. By using a newly developed Congo red assay and an ELISA test, we demonstrated that the TTSS-targeted activity of these polyphenolic extracts also affects the TTSS pilus assembly. In consideration of the potential application of polyphenolic extracts in plant protection, the absence of any toxicity of these polyphenolic compounds was also assessed. A widely and evolutionary conserved molecular target such as Ca2+-ATPase, essential for the survival of any living organism, was used for the toxicity assessment.