Giulio Dimaria

Ciclo: XXXV

Data inizio: 31/10/2019

Curriculum: Agroalimentari

Borsa: UniCT

Titolo tesi: Pseudomonas interactions in the plant microbiome and role in the biocontrol of Plenodomus tracheiphilus causal agent of Citrus Mal secco disease

Abstract: Mal secco disease (MSD) is a severe citrus tracheomycosis caused by the mitosporic fungus Plenodomus tracheiphilus (Pt). In this study we examined the effect of MSD infection on root (rhizosphere and endorhizosphere) and xylem endosphere microbial communities of Citrus aurantium (sour orange) seedlings following leaf- and root- inoculation with Pt through a metagenomic analysis based on 16S rDNA and ITS amplicons sequencing. The pathogen was detected by real-time PCR in the roots of root-inoculated plants and in the xylem of both leaf- and root-inoculated plants. Our results suggest that leaf and root infection with Pt alters the bacterial and fungal community composition of the compartments analyzed through a decrease or increase in the abundance of some key taxa. The genus Pseudomonas, which includes strains with known biocontrol activity, was the first taxon represented in the rhizosphere and the second in the endorhizosphere of both inoculated and uninoculated plants. According to differential abundance analysis, bacterial populations of this genus were significantly reduced in the roots of seedlings inoculated with the pathogen. The analysis of co-occurrence networks also suggested, overall, a change in the structure of microbial communities following infection with Pt.

The P. corrugata subclade (P. corrugata, P. mediterranea, P. brassicacearum, P. thivervalensis, P. kilonensis) has plant growth promotion and biocontrol activities against various phytopathogens. A collection of P. corrugata and P. mediterranea strains from the Phytobacteriology laboratory (Di3A) was screened in vitro for biocontrol activity against different isolates of P. tracheiphilus. All bacterial strains tested showed in vitro antagonistic activity, albeit to varying degrees. The two most effective biocontrol strains in vitro, P. corrugata PVCT 4.3t and P. mediterranea PVCT 3C, were selected for further in vivo antagonistic assays and whole genome sequencing (WGS). Both strains significantly reduced the incidence and severity of MSD in lemon and sour orange plants especially in the early stages of infection. To investigate genes potentially involved in biocontrol activity, total genomic DNA from both strains was extracted and sequenced using the Illumina NovaSeq PE150 platform. Based on sequencing output, the genomes of P. corrugata PVCT 4.3t and P. mediterranea PVCT 3C harbor numerous biosynthetic gene clusters mainly coding for non-ribosomal peptide synthetases (NRPS) involved in the production of antimicrobial compounds. Collectively, these results indicate that the two selected strains are effective biological control agents against P. tracheiphilus both in vitro and in vivo.

Based on the results obtained from genome analysis, we also studied the biological role of two genes (crmA, solR), never investigated before, in the production of secondary metabolites using the model strain CFBP 5454 of P. corrugata. P. corrugata possesses a canonical quorum sensing (QS) system, PcoI/PcoR, based on the signal molecules acyl-homoserine-lactones (AHL), and in this thesis project we focused on the role of a transcriptional regulator belonging to the LuxR solos family designated SolR. P. corrugata produces the cyclic lipopeptides (CLP) cormycin A and corpeptins A and B. The contribution of cormycin A has been investigated here for the first time. The PCOSOLR and PCOCRMA mutant strains were obtained by inactivating, respectively, solR and crmA genes (the latter coding for a non-ribosomal peptide synthase) of P. corrugata CFBP 5454 and the resulting mutational phenotypes were studied. PCOSOLR mutant strain was able to induce hypersensitive response (HR) in the non-host plant tobacco and was more virulent in tomato, probably due to an increased production of CLP with phytotoxic activity. Instead, phenotype analysis of PCOCRMA mutant strain suggested that this strain produces fewer antimicrobial compounds than the wild-type strain. PCOCRMA was in fact unable to cause HR in tobacco and to inhibit the growth of some microorganisms sensitive to CLP, while the symptoms of pith necrosis in tomato were significantly reduced. Based on these results, SolR and CrmA play an important role in the interactions of P. corrugata with plants, in virulence, in broad-spectrum antimicrobial activity and, presumably, in the ability of this bacterium to colonize new ecological niches.

Tutor: Catara

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Email: dimaria_giulio@libero.it

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Periodi all'estero- Sede e data: International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy (from 18/05/2021 to 20/07/2021 and from 18/10/2021 to 21/12/2021)

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