Aureobasidium pullulans as biological control agent: modes of action

The postharvest phase has been considered an environment very suitable for successful application of biological control agents (BCAs). However, the tri-interaction between fungal pathogen, host (fruit) and antagonist is influenced by several parameters such as temperature, oxidative stresses, oxygen composition, water activity, etc. that could be determining for the success of biocontrol. Knowledge of the modes of action of BCAs is essential in order to enhance their viability and increase their potentialities in disease control. The thesis focused on the possibility to explain the modes of action of a biological control agent (BCA): Aureobasidium pullulans, in particular the strains L1 and L8, control effective against fruit postharvest fungal pathogen. In particular in this work were studied the different modes of action of BCA, such as: i) the ability to produce volatile organic compounds (VOCs), identified by SPME- gas chromatography-mass spectrometry (GC-MS) and tested by in vitro and in vivo assays against Penicillium spp., Botrytis cinerea, Colletotrichum acutatum; ii) the ability to produce lytic enzymes (exo and endo chitinase and β-1,3-glucanase) tested against Monilinia laxa, causal agent of brown rot of stone fruits. L1 and L8 lytic enzymes were also evaluated through their relative genes by molecular tools; iii) the competition for space and nutrients, such as sugars (sucrose, glucose and fructose) and iron; the latter induced the production of siderophores, molecules with high affinity for iron chelation. A molecular investigation was carried out to better understand the gene regulation strictly correlated to the production of these chelating molucules. The competition for space against M. laxa was verified by electron microscopy techniques; iv) a depth bibliographical analysis on BCAs mechanisms of action and their possible combination with physical and chemical treatments was conducted.

Studies of sustainable strategies to control phytopathogen agents

Choosing natural enemies to suppress pest population has been for a long the key of biological control. Overtime the term biological control has also been applied to the use of suppressive soils, bio-disinfection and biopesticides. Biological control agents (BCA) and natural compounds, extracted or fermented from various sources, are the resources for containing phytopathogens. BCA can act through direct antagonism mechanisms or inducing hypovirulence of the pathogen. The first part of the thesis focused on mycoviruses infecting phytopathogenic fungi belonging to the genus Fusarium. The development of new approaches capable of faster dissecting the virome of filamentous fungi samples was performed. The semiconductor-based sequencer Ion Torrent™ and the nanopore-based sequencer MinION have been exploited to analyze DNA and RNA referable to viral genomes. Comparison with GeneBank accessions and sequence analysis allowed to identify more than 40 putative viral species, some of these mycovirus genera have been studied as inducers of hypovirulence in several phytopathogenic fungi, therefore future works will focus on the comparison of the morphology and physiology of the fungal strain infected and cured by the viruses identified and their possible use as a biocontrol agent. In a second part of the thesis the potential of botanical pesticides has been evaluated for the biocontrol of phloem limited phytopathogens such as phytoplasmas. The only active compounds able to control phytoplasmas are the antibiotic oxytetracyclines and in vitro direct and fast screening of new antimicrobials compounds on media is almost impossible due to the difficulty to culture phytoplasmas. For this reason, a simple and reliable screening method was developed to evaluate the effects of antimicrobials directly on phytoplasmas by an “ex-vivo” approach. Using scanning electron microscopy (SEM) in parallel with molecular tools (ddRT-PCR), the direct activity of tetracyclines on phytoplasma cells was verified, identifying also a promising compound showing similar activity.

Unraveling the pathophysiological mechanisms of visceral pain in the murine model of Fabry disease

Fabry disease (FD) is an X‐linked inherited, lysosomal storage disorder characterized by a deficient activity of the enzyme α-Galactosidase A (α-Gal A). This deficiency causes an accumulation of globotriaosylceramide 3 (Gb3), in nearly all organs. Gastrointestinal (GI) symptoms are among the earliest and most frequent symptoms of FD. It has been hypothesized that Gb3 accumulation is the leading cause of these, but their pathophysiology is complex and still poorly understood. Here, we aim at understanding the molecular mechanisms underpinning the GI symptoms of FD. For this purpose, we used the α‐Gal A (-/0) male mouse, a murine model of FD, to characterize morphological and molecular features of the colon tract. Our results show that α‐Gal A (-/0) mice display a thickening of the muscular layer due to a hypertrophic state of myenteric plexus ganglia, caused by an accumulation of Gb3 in neurons. Also, α-Gal A (-/0) mice present a decreased density of mucosal nerve fibres. Furthermore, α-Gal A (-/0) mice presented visceral hyperalgesia, by showing greater visceromotor response (VMR) values and obtaining higher abdominal withdrawal reflex (AWR) scores, following colorectal distension (CRD). Subsequently, the immunoreactivity of the pain-related ion channels TRPV1, TRPV4, TRPA1 and TRPM8 was detected at level of myenteric and submucosal plexus ganglia of both the genotypes. Further studies are required to assess differences of expression between α-Gal A (-/0) and control mice. Finally, we optimized the protocols to obtain three types of primary cultures from mouse intestine to be tested electrophysiologically: a mixed culture containing neurons and glia, an enriched culture of neurons, and one of glia. In summary, we revealed alterations that are likely to be part of the pathophysiological causes of FD GI symptoms. Therefore, together with further studies, this work could help identify new therapeutic targets for the treatment of visceral pain in FD.