Fusarium head blight: characterization of secondary Fusarium species, evaluation of their pathogenetic role and ability to produce mycotoxins

Fusarium head blight (FHB) is a worldwide cereal disease caused by a complex of Fusarium species resulting in high yield losses, reduction in quality and mycotoxin contamination of grain. A shift in Fusarium head blight community has been observed worldwide. The present work aimed to analyze the evolution of Italian FHB community focusing the attention on species considered “secondary” in the past years such as members of Fusarium tricinctum species complex (FTSC) and F. proliferatum. The first goal of the study was to analyze the fungal community associated with Italian durum wheat in two different years. F. poae, F. avenaceum and F. proliferatum were the main species detected on Italian durum kernels. A variable mycotoxins contamination was observed in the analyzed samples. Considering, the increased incidence of F. avenaceum and other members of FTSC in Italian FHB, the second aim was to investigate genetic diversity among the FTSC and estimate the mycotoxin risk related to these species. Phylogenetic analyses revealed that F. avenaceum (FTSC 4) was the most common species in Italy, followed by an unnamed Fusarium sp., F. tricinctum and F. acuminatum. In addition to these four phylospecies, five other F. tricinctum clade species were sampled. These included strains of four newly discovered species (Fusarium spp. FTSC 11, 13, 14, 15) and F. iranicum (FTSC 6). Most isolates tested for mycotoxin production on rice cultures were able to produce quantitative levels of enniatins and moniliformin. In addition, a preliminary study was conducted to evaluate the ability of a selected F. proliferatum isolate to produce fumonisins on wheat in open field and under natural climatic conditions. The three analogues (FB1, FB2 and FB3) were quantified by HPLC-FLD analysis on kernels, chaff and rachis. Fumonisins were detected in all the three investigated fractions without significant differences.

Novel hydroxystearoyl-, heterocyclic and carbocyclic derivatives: synthesis and applications in biological field

This PhD project has been mainly focused on the synthesis of novel organic compounds containing heterocyclic and/or carbocyclic scaffold and on the study of stearic acid derivatives and their applications in biological field. The synthesis of novel derivatives of 9-hydroxystearic acid (9-HSA) evidenced how the presence of substituents on C9, able to make hydrogen bonds is of crucial importance for the biological activity. Also the position of the hydroxy group along the chain of hydroxystearic acids was investigated: regioisomers with the hydroxy group bound to odd carbons resulted more active than those bearing the hydroxy group on even carbons. Further, the insertion of (R)-9-HSA in magnetic nanoparticles gave a novel material which characterization remarked its suitability for drug delivery. Structural hybrids between amino aza-heterocycles and azelaic acid have been synthesized and some of them showed a selective activity towards osteosarcoma cell line U2OS. Several Apcin analogues bearing indole, benzothiazole, benzofurazan moieties connected to tryptaminyl-, amino pyridinyl-, pyrimidinyl- and pyrazinyl ring through a 1,1,1-trichloroethyl group were synthesized. Biological tests showed the importance of both the tryptaminyl and the pyrimidinyl moieties, confirming the effectiveness against acute leukemia models. The SNAr between 2-aminothiazole derivatives and 7-chlorodinitrobenzofuroxan revealed different behaviour depending from amino substituent of the thiazole. The reaction with 2-N-piperidinyl-, 2-N-morpholinyl-, or 2-N-pyrrolidinyl thiazole gave two isomeric species derived from the attack on C-5 of thiazole ring. Thiazoles substituted with primary- or not-cyclic secondary amines reacted with the exocyclic amino nitrogen atom giving a series of compounds whose biological activity have highlighted as they might be promising candidates for further development of antitumor agents. A series of 9-fluorenylidene derivatives, of interest in medical and optoelectronic field as organic scintillators, was synthesized through Wittig or Suzuky reaction and will be analyzed to test their potential scintillatory properties.

Insights on epidemiology and control of Marek’s disease through traditional and novel molecular tools

Marek's disease (MD) is a contagious, lymphoproliferative and neuropathic disease of poultry caused by a ubiquitous lymphotropic and oncogenic virus, Gallid alphaherpesvirus 2 (GaHV-2). MD has been reported in all poultry-rearing countries and is among the viral diseases with the highest economic impact in the poultry industry worldwide, including Italy. MD has been also recognized as one of the leading causes of mortality in backyard poultry. The present doctoral thesis aimed at exploring Marek's disease virus molecular epidemiology in Italian commercial and backyard chicken flocks and, for the first time, in commercial turkeys affected by clinical MD. Molecular biology techniques targeting the full-length meq gene, the major GaHV-2 oncogene, were used to detect and characterize the circulating GaHV-2 strains searching for genetic markers of virulence. A final study focused on the development of rapid, sensitive, and species-specific loop-mediated isothermal amplification assays coupled with a lateral flow device readout for the detection of conventional and recombinant HVT-based vaccines is included in the thesis. HVT vaccines, currently used to protect chickens from MD, are referred to as "leaky", as they do not impede the infection, replication, and shedding of field GaHV-2: vaccinal and field viruses can coexist in the vaccinated host and molecular tests able to discriminate between GaHV-2 and HVT are required. These new simple, fast, and accurate tests for the monitoring of MD vaccination success in the field could be greatly beneficial for field veterinarians, small laboratories, and more broadly for resource-limited settings.

Monitoring of Mediterranean marine benthic biodiversity through novel, standardized, and integrated approaches

Marine healthy ecosystems support life on Earth and human well-being thanks to their biodiversity, which is proven to decline mainly due to anthropogenic stressors. Monitoring how marine biodiversity changes trough space and time is needed to properly define and enroll effective actions towards habitat conservation and preservation. This is particularly needed in those areas that are very rich in species compared to their low surface extension and are characterized by strong anthropic pressures, such as the Mediterranean Sea. Subtidal rocky benthic Mediterranean habitats have a complex structural architecture, hosting a panoply of tiny organisms (cryptofauna) that inhabit crevices and caves, but that are still unknown. Different artificial standardized sampling structures (SSS) and methods have been developed and employed to characterize the cryptofauna, allowing for data replicability and comparability across regions. Organisms growing on these artificial structures can be identified coupling morphological taxonomy and DNA barcoding and metabarcoding. The metabarcoding allows for the identification of organisms in a bulk sample without morphological analysis, and it is based on comparing the genetic similarities of the assessed organisms with barcoding sequences present in online barcoding repositories. Nevertheless, barcoded species nowadays represent only a small portion of known species, and barcoding reference databases are not always curated and updated on a regular basis. In this Thesis I used an integrative approach to characterize benthic marine biodiversity, specifically coupling morphological and molecular techniques with the employment of SSS. Moreover, I upgraded the actual status of COI (cytochrome c oxidase subunit I) barcoding of marine metazoans, and I built a customized COI barcoding reference database for metabarcoding studies on temperate biogenic reefs. This work implemented the knowledge about diversity of Mediterranean marine communities, laying the groundworks for monitoring marine and environmental changes that will occur in the next future as consequences of anthropic and climate threats.

Identification of resistance factors to the main wheat diseases in the Mediterranean area

Several diseases challenge bread and durum wheat productions worldwide. The importance of these cereals requires adequate protection to pathogens that can cause strong yield and grain quality losses. The main work of this thesis was related to phenotype GDP (Global Durum Panel) in the Mediterranean region (Italy, Egypt, Lebanon, Morocco and Turkey) and Argentina across three years (2019-2021) for yellow rust resistance (infection type and severity). GWAS shows in particular, loci in chromosome 1B, 2B, 4B, 5A, 6A, 7B showed high significance across nurseries/years, with various patterns of GxE. The second chapter is about Zymoseptoria tritici, agent of STB (Septoria Tritici Blotch), a foliar pathogen that yearly causes high damages if not controlled. In recent years research in durum wheat breeding is focused on the identification of novel, underexploited resistance genes to be subsequently and conveniently moved into the pre-breeding and breeding stream. The plants were phenotyped for disease height characters, infection type at the flag leaf and infection type at the level of the canopy below the flag leaf. This experiment opens up a rich scenario of analysis and opportunities to investigate and discover new loci of resistance to STB. Third chapter is about Fusarium head blight (FHB) is a fungal disease caused by pathogens belonging to the genus Fusarium. In particular, Fusarium culmorum and Fusarium graminearum species cause severe grain yield losses and accumulation of mycotoxins in wheat that compromise food safety. Over 250 QTL/genes for FHB resistance have been identified in bread wheat, such as Fhb 1 and Fhb 5 but only a small number of FHB resistance loci have been mapped in durum wheat. The aim of this work is to find loci of partial resistance to FHB already present in durum and bread wheat germplasm and therefore easily cumulative.

Novel functionalized calcium phosphates: structures, morphologies and potential applications.

The stable increase in average life expectancy and the consecutive increase in the number of cases of bone related diseases has led to a growing interest in the development of materials that can promote bone repair and/or replacement. Among the best candidates are those materials that have a high similarity to bones, in terms of composition, structure, morphology and functionality. Biomineralized tissue, and thus also bones, have three main components: water, an organic matrix and an inorganic deposit. In vertebrates, the inorganic deposit consists of what is called biological apatite, which slightly differ from stoichiometric hydroxyapatite (HA) both in crystallographic terms and in the presence of foreign atoms and species. This justifies the great attention towards calcium phosphates, which show excellent biocompatibility and bioactivity. The performances of the material and the response of the biological tissue can be further improved through their functionalization with ions, biologically active molecules and nanostructures. This thesis focuses on several possible functionalizations of calcium phosphates, and their effects on chemical properties and biological performances. In particular, the functionalizing agents include several biologically relevant ions, such as Cobalt (Co), Manganese (Mn), Strontium (Sr) and Zinc (Zn); two organic molecules, a flavonoid (Quercetin) and a polyphenol (Curcumin); and nanoparticles, namely tungsten oxide (WO3) NPs. Functionalization was carried out on various calcium phosphates: dicalcium phosphate dihydrate (DCPD), dicalcium phosphate anhydrous (DCPA) and hydroxyapatite (HA). Two different strategies of functionalization were applied: direct synthesis and adsorption from solution. Finally, a chapter is devoted to a preliminary study on the development of cements based on some of the functionalized phosphates obtained.

Acoustic backscatter of small pelagic fish species in the Adriatic Sea

The aim of the present study is to apply a broad range of techniques to increase the knowledge of acoustic properties of Sprattus sprattus, Scomber colias and Trachurus mediterraneus in the Adriatic Sea. A novel study using tethered live fish but not involving hooks and anesthetic was tested on T. mediterraneus and S. colias through several ex situ experiments using a split-beam scientific echosounder operating at 38, 120, and 200 kHz. The mean TS was estimated for 29 live specimens, resulting in a conversion factor b20 value of -71.4 dB re 1 m2 and -71.6 dB re 1 m2 respectively which is ~3 dB lower than the current one in use in the Mediterranean Sea. Successively, two monospecific trawl hauls were analyzed through the application of in situ approach for the computation of TS values of S. sprattus which led to six b20 values for sprat (range, -68.8 dB re 1 m2 to -65.6 dB re 1 m2), all higher than the current known value of -71.7 dB re 1 m2. The high difference up to 4.2 dB compared to the current value translates in a significant decrease of absolute sprat biomass along the time series un to 20%. Finally, 149 specimens of the three species were collected for backscattering model application(i.e. Kirchhoff-ray mode model (KRM) and Finite Element Method (FEM)) from digital images of the fish body and swimbladder obtained from Computer Tomography (CT) and X-Ray scans. The values resulting from the application of KRM and FEM are in agreement with empirical results. In general terms the present work proposes the acoustic backscatter characterization of S. colias, S. sprattus and T. mediterraneus in the Mediterranean Sea.

Investigation of plant genetic-microbiome interactions in different plant species

The rhizosphere, i.e. the soil surrounding the plant roots, and endosphere, i.e. the microbial communities within the plant organs harbors microbes known to influence root and plant physiological processes. An important question is to what extent plant species, genotypes and environmental conditions affect bacterial and fungal communities. The objectives of the first research study were to unravel and compare the rhizospheric microbiota of grape in two independent vineyards using 16S and ITS amplicon sequencing, evaluate location and varietal effects, and test the correlation between bioavailable copper levels and other soil parameters with microbiota composition and diversity. Our results showed that the microbial alpha diversity based on Shannon index differed significantly between vineyards while it did not differ between two grape cultivars. In the second study, we were focusing on different wheat species and genotypes such as Bread Wheat, Wild Emmer Wheat, Domesticated Emmer Wheat, Durum Wheat Landraces, Durum Wheat cultivars, T. monococcum and triticale in two fields located in Bologna and Foggia. The objectives of this research experiment were to elucidate and compare the rhizospheric and endophytic microbiota of 30 diverse wheat genotypes in two different fields using 16S amplicon sequencing. Our results showed that the microbial alpha diversity based on Shannon index differed significantly between fields of Bologna and Foggia, in which Bologna had a higher diversity in respect to Foggia for both rhizospheric and endophytic communities. Using Shannon index there was significant differences, for instance, between Durum Emmer Wheat and Wild Emmer Wheat in Bologna, and between Bread Wheat and Durum Wheat Landraces in Foggia. Our results contribute to understand the role of wheat species and genotype and the filed management on the root-microbe-soil interactions in the perspective of understanding their impact on crop systems sustainability.

Desquamation Is A Novel Phenomenon For Collective Prostate Epithelial Cell Deletion After Castration.

The mechanism underlying castration-induced prostate regression, which is a classical physiological concept translated into the therapeutic treatment of advanced prostate cancer, involves epithelial cell apoptosis. In searching for events and mechanisms contributing to prostate regression in response to androgen modulation, we have frequently observed the collective deletion of epithelial cells. This work was undertaken to characterize this phenomenon hereafter named desquamation and to verify its presence after 17β-estradiol (E2) administration. Electron microscopy revealed that the desquamating cells had preserved cell-cell junctions and collapsed nuclear contents. The TUNEL reaction was negative for these cells, which were also negative for cleaved caspases-8, -9, -3 and nuclear apoptosis-inducing factor. Detailed analyses revealed that the condensed chromatin was first affected detaching from the nuclear lamina, which was observable after lamin A immunohistochemistry, suggesting the lack of lamin A degradation. A search in animals treated with supraphysiological E2 employed as an alternative anti-androgen treatment revealed no desquamation. The combined treatment (Cas + E2 group) caused changes particular to each treatment, including desquamation. In conclusion, desquamation appeared as a novel phenomenon contributing to collective prostate epithelial cell deletion, distinct from the classical castration-induced apoptosis and particular to the androgen deprivation resulting from surgical castration, and should be considered as part of the mechanisms promoting organ regression.

Mucopolysaccharidosis Type Ii: Identification Of 30 Novel Mutations Among Latin American Patients.

In this study, 103 unrelated South-American patients with mucopolysaccharidosis type II (MPS II) were investigated aiming at the identification of iduronate-2-sulfatase (IDS) disease causing mutations and the possibility of some insights on the genotype-phenotype correlation The strategy used for genotyping involved the identification of the previously reported inversion/disruption of the IDS gene by PCR and screening for other mutations by PCR/SSCP. The exons with altered mobility on SSCP were sequenced, as well as all the exons of patients with no SSCP alteration. By using this strategy, we were able to find the pathogenic mutation in all patients. Alterations such as inversion/disruption and partial/total deletions of the IDS gene were found in 20/103 (19%) patients. Small insertions/deletions/indels (<22 bp) and point mutations were identified in 83/103 (88%) patients, including 30 novel mutations; except for a higher frequency of small duplications in relation to small deletions, the frequencies of major and minor alterations found in our sample are in accordance with those described in the literature.

Evaluation Of Different Treatment Methods Against Denture Stomatitis: A Randomized Clinical Study.

The aim of this clinical study was to determine the efficacy of Uncaria tomentosa (cat's claw) against denture stomatitis (DS). Fifty patients with DS were randomly assigned into 3 groups to receive 2% miconazole, placebo, or 2% U tomentosa gel. DS level was recorded immediately, after 1 week of treatment, and 1 week after treatment. The clinical effectiveness of each treatment was measured using Newton's criteria. Mycologic samples from palatal mucosa and prosthesis were obtained to determinate colony forming units per milliliter (CFU/mL) and fungal identification at each evaluation period. Candida species were identified with HiCrome Candida and API 20C AUX biochemical test. DS severity decreased in all groups (P < .05). A significant reduction in number of CFU/mL after 1 week (P < .05) was observed for all groups and remained after 14 days (P > .05). C albicans was the most prevalent microorganism before treatment, followed by C tropicalis, C glabrata, and C krusei, regardless of the group and time evaluated. U tomentosa gel had the same effect as 2% miconazole gel. U tomentosa gel is an effective topical adjuvant treatment for denture stomatitis.

Development And Characterization Of Microsatellite Loci For Ocotea Species (lauraceae) Threatened With Extinction.

The Atlantic rainforest species Ocotea catharinensis, Ocotea odorifera, and Ocotea porosa have been extensively harvested in the past for timber and oil extraction and are currently listed as threatened due to overexploitation. To investigate the genetic diversity and population structure of these species, we developed 8 polymorphic microsatellite markers for O. odorifera from an enriched microsatellite library by using 2 dinucleotide repeats. The microsatellite markers were tested for cross-amplification in O. catharinensis and O. porosa. The average number of alleles per locus was 10.2, considering all loci over 2 populations of O. odorifera. Observed and expected heterozygosities for O. odorifera ranged from 0.39 to 0.93 and 0.41 to 0.92 across populations, respectively. Cross-amplification of all loci was successfully observed in O. catharinensis and O. porosa except 1 locus that was found to lack polymorphism in O. porosa. Combined probabilities of identity in the studied Ocotea species were very low ranging from 1.0 x 10-24 to 7.7 x 10-24. The probability of exclusion over all loci estimated for O. odorifera indicated a 99.9% chance of correctly excluding a random nonparent individual. The microsatellite markers described in this study have high information content and will be useful for further investigations on genetic diversity within these species and for subsequent conservation purposes.

Developmental Rate Of Immatures Of Two Fly Species Of Forensic Importance: Sarcophaga (liopygia) Ruficornis And Microcerella Halli (diptera: Sarcophagidae).

Since insect species are poikilothermic organisms, they generally exhibit different growth patterns depending on the temperature at which they develop. This factor is important in forensic entomology, especially for estimating postmortem interval (PMI) when it is based on the developmental time of the insects reared in decomposing bodies. This study aimed to estimate the rates of development, viability, and survival of immatures of Sarcophaga (Liopygia) ruficornis (Fabricius 1794) and Microcerella halli (Engel 1931) (Diptera: Sarcophagidae) reared in different temperatures: 10, 15, 20, 25, 30, and 35 ± 1 °C. Bovine raw ground meat was offered as food for all experimental groups, each consisting of four replicates, in the proportion of 2 g/larva. To measure the evolution of growth, ten specimens of each group were randomly chosen and weighed every 12 h, from initial feeding larva to pupae, and then discarded. Considering the records of weight gain, survival rates, and stability of growth rates, the range of optimum temperature for the development of S. (L.) ruficornis is between 20 and 35 °C, and that of M. halli is between 20 and 25 °C. For both species, the longest times of development were in the lowest temperatures. The survival rate at extreme temperatures (10 and 35 °C) was lower in both species. Biological data such as the ones obtained in this study are of great importance to achieve a more accurate estimate of the PMI.

Molecular And Morphological Evidence Reveals A New Species In The Phyllomedusa Hypochondrialis Group (hylidae, Phyllomedusinae) From The Atlantic Forest Of The Highlands Of Southern Brazil.

The taxonomic status of a disjunctive population of Phyllomedusa from southern Brazil was diagnosed using molecular, chromosomal, and morphological approaches, which resulted in the recognition of a new species of the P. hypochondrialis group. Here, we describe P. rustica sp. n. from the Atlantic Forest biome, found in natural highland grassland formations on a plateau in the south of Brazil. Phylogenetic inferences placed P. rustica sp. n. in a subclade that includes P. rhodei + all the highland species of the clade. Chromosomal morphology is conservative, supporting the inference of homologies among the karyotypes of the species of this genus. Phyllomedusa rustica is apparently restricted to its type-locality, and we discuss the potential impact on the strategies applied to the conservation of the natural grassland formations found within the Brazilian Atlantic Forest biome in southern Brazil. We suggest that conservation strategies should be modified to guarantee the preservation of this species.

Comparative Cytogenetics Of Physalaemus albifrons And Physalaemus Cuvieri Species Groups (anura, leptodactylidae).

Recently, Physalaemus albifrons (Spix, 1824) was relocated from the Physalaemus cuvieri group to the same group as Physalaemus biligonigerus (Cope, 1861), Physalaemus marmoratus (Reinhardt & Lütken, 1862) and Physalaemus santafecinus Barrio, 1965. To contribute to the analysis of this proposition, we studied the karyotypes of Physalaemus albifrons, Physalaemus santafecinus and three species of the Physalaemus cuvieri group. The karyotype of Physalaemus santafecinus was found to be very similar to those of Physalaemus biligonigerus and Physalaemus marmoratus, which were previously described. A remarkable characteristic that these three species share is a conspicuous C-band that extends from the pericentromeric region almost to the telomere in the short arm of chromosome 3. This characteristic is not present in the Physalaemus albifrons karyotype and could be a synapomorphy of Physalaemus biligonigerus, Physalaemus marmoratus and Physalaemus santafecinus. The karyotype of Physalaemus santafecinus is also similar to those of Physalaemus marmoratus and Physalaemus biligonigerus owing to the presence of several terminal C-bands and the distal localization of the NOR in a small metacentric chromosome. In contrast, the Physalaemus albifrons karyotype has no terminal C-bands and its NOR is located interstitially in the long arm of submetacentric chromosome 8. The NOR-bearing chromosome of Physalaemus albifrons very closely resembles those found in Physalaemus albonotatus (Steindachner, 1864), Physalaemus cuqui Lobo, 1993 and some populations of Physalaemus cuvieri Fitzinger, 1826. Additionally, the Physalaemus albifrons karyotype has an interstitial C-band in chromosome 5 that has been exclusively observed in species of the Physalaemus cuvieri group. Therefore, we were not able to identify any chromosomal feature that supports the reallocation of Physalaemus albifrons.