Molecular methods for the diagnosis and characterization of phytopathogenic fungi of quarantine concern or causing emerging plant diseases

In the context of molecular techniques applied to Plant Pathology, this Ph.D. thesis has pursued the following major objectives: i) to develop new diagnostic protocols for fungal pathogens; ii) to study the metabolic and physiological effects determined by new and emerging fungal pathogens; iii) to contribute to develop management strategies of diseases caused by quarantine and/or emerging fungi on plant species typical of the Mediterranean region. To fulfill these objectives, the following specific studies have been developed: • “Fusarium circinatum an emergent and quarantine pathogen of pine worldwide: its detection and its interaction with Phytophthora species (P. cambivora and P. parvispora) on Pinus radiata seedlings.” This study has been developed by two different lines of research: “Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen, Fusarium circinatum.” In this study, different protocols for the molecular diagnosis of F. circinatum were compared and validated for the first time in 23 laboratories spread across Europe, South Africa and Chile, in the framework of the international collaborative study funded by COST Action FP1406 “Pine pitch canker - Strategies for management of Gibberella circinata in greenhouses and forests - PINESTRENGTH ''. The protocol tested by the Laboratory of Molecular Plant Pathology of the Department of Agriculture, Food and Environment (Di3A) of the University of Catania were: (i) Real-time PCR by Lamarche et al. 2015 and (ii) real-time PCR by Luchi et al. 2018. Results from the two tested protocols were illustrated in Ioos et al (2019), Scientific Reports 2019, 9, 8195. DOI: 10.1038 / s41598-019-44672-8. “Co-infections by Fusarium circinatum and Phytophthora spp. on Pinus radiata, a case study of complex interactions in the Pine pitch canker disease.” This study investigated i. the phenotypic response of pine to the infective process and ii. the relative expression levels of genes of plant encoding for pathogenetic-related proteins and antifungal secondary metabolites. Results obtained in this research showed that the phenotypic response of pine to the simultaneous action of the aforementioned pathogens it is manifested by an increasing of the severity of the symptoms at the early stages of the infection, allowing then to speculate that Phytophthora spp. can realistically contribute to the severity of the disease. Results from gene expression suggest that a real synergic effect as the result of the effects of both pathogens it is not clearly evident. • “Scabby canker caused by Neofusicoccum batangarum (Botryosphaeriaceae), an emergent disease of Opuntia ficus-indica in minor islands around Sicily: identification of the causal agent and characterization of both its phytotoxic metabolites and the genetic variability of its local population.” The specific objectives of this study were the following: i) determine the geographical distribution of the disease; ii) characterize N. batangarum isolates obtained from symptomatic plants of prickly pear in the smaller islands of Sicily; iii) check whether the range of potential host plants of this phytopathogenic fungus includes other Mediterranean species that could act as alternative hosts or as inoculation 'reservoir'; iv) determine the ability of N. batangarum to produce phytotoxic secondary metabolites (phytotoxins) in culture, which can play an active role in the pathogenesis of the disease; v) chemically identify the phytotoxins extracted from N. batangarum liquid filtrates and determine their phytotoxic effects on the host plant as well as on non-host plant species. In cross-pathogenicity tests, N. batangarum isolated from Opuntia ficus-indica plants was able to reproduce disease symptoms on the host plant and also infect other plant species. The fungus artificially inoculated by wounding induced cancers on several hosts. This result indicates that the pathogen has a very wide range of potential hosts. Six phytotoxins were obtained and identified from Neofusicoccum batangarum culture filtrates: (-) - (R) ‐mellein (1); (±) ‐botriisocoumarin A (2); (-) - (3R, 4R) - and (-) - (3R, 4S) ‐4 hydroxymelline (3 and 4); (-) - terpestacin (5); and (+) - 3,4 ‐ dihydro - 4,5,8 - trihydroxy - 3 - methylisocoumarin, renamed (+) - neoisocoumarin (6). All six metabolites have been shown to have phytotoxic activity on both the host and non-host plants. The most active compounds proved to be (±) ‐botriisocoumarin A (2), (-) - terpestacin (5) and (+) - neoisocoumarin (6). Results from this study are part of two scientific publications: Masi, Mt. et al., Toxins 2020, 12, 126. DOI: 10.3390 / toxins12020126 and Aloi, F. et al., Mediterranean Phytopathology 2020, 59 (2): 269-284. DOI: 10.14601 / Phyto-11225. • “Identification of Neofusicoccum parvum (Botryosphaeriaceae) as the causative agent of gummy cankers of lemon (Citrus × limon) trees.” This study was aimed at identifying the causative agent of the observed disease. Neofusicoccum parvum, in the family Botryosphaeriaceae, was identified as the causal agent of bot gummosis of lemon (Citrus x limon) trees, in the two major lemon-producing areas in Italy. Gummy cankers on trunk and scaffold branches of mature trees were the most typical disease symptoms. Neofusicoccum parvum was the sole fungus constantly and consistently isolated from the canker bark of symptomatic lemon trees. It was identified on the basis of morphological characters and the phylogenetic analysis of three loci, i. e. the internal transcribed spacer of nuclear ribosomal DNA (ITS) as well as the translation elongation factor 1-alpha (TEF1) and β-tubulin (TUB2) genes. The pathogenicity of N. parvum was demonstrated by wound inoculating two lemon cultivars, ‘Femminello 2kr’ and ‘Monachello’, as well as citrange (C. sinensis x Poncirus trifoliata) ‘Carrizo’ rootstock. In artificial inoculations, the fungus was very aggressive on lemons and weakly virulent on citrange, consistently with symptoms observed in the field as a consequence of natural infections. This is the first report of N. parvum, both in a wide and in a strict taxonomic sense, as a pathogen of lemon in Italy. • “Characterization of Alternaria species associated with heart rot of pomegranate fruit.” This study was aimed at identifying Alternaria species associated with heart rot disease of pomegranate fruit in southern Italy and characterizing their mycotoxigenic profile. A total of 42 Alternaria isolates were characterized. They were obtained from pomegranate fruits with symptom of heart rot sampled in Apulia and Sicily and grouped into six distinct morphotypes based on macro- and microscopic features. According to multi-gene phylogenetic analysis, including internal transcribed spacer (ITS), translation elongation factor 1-α (EF-1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a SCAR marker (OPA10-2), 38 isolates of morphotypes 1 to 5 were identified as A. alternata, while isolates of morphotype 6, all from Sicily, clustered within the A. arborescens species complex. In particular, isolates of morphotype 1, the most numerous, clustered with the ex-type isolate of A. alternata, proving to belong to morphotype alternata. No difference in pathogenicity on pomegranate fruits was found between isolates of A. alternata and A. arborescens and among A. alternata isolates of different morphotypes. The toxigenic profile of isolates varied greatly: in vitro, all 42 isolates produced tenuazonic acid and most of them other mycotoxins including alternariol, alternariol monomethyl ether, altenuene and tentoxin. • “Shoot dieback of citrus, a new disease caused by Colletotrichum species.” This study was aimed at identifying the Colletotrichum species associated with twig and shoot dieback of citrus, a new disease occurring in the Mediterranean region and also reported as emerging in California. Overall, 119 Colletotrichum isolates were characterized. They were recovered from symptomatic trees of sweet orange, mandarin and mandarin-like during a survey of citrus groves in Albania and Sicily (southern Italy). The isolates were grouped into two distinct morphotypes. The grouping of isolates was supported by phylogenetic sequence analysis of two genetic markers, the internal transcribed spacer regions of rDNA (ITS) and β-tubulin (TUB2). The groups were identified as Colletotrichum gloeosporioides and C. karstii, respectively. The former accounted for more than 91% of isolates, while the latter was retrieved only occasionally in Sicily. Both species induced symptoms on artificially wound inoculated twigs. C. gloeosporioides was more aggressive than of C. karstii. Winds and prolonged drought were the factor predisposing to Colletotrichum twig and shoot dieback. This is the first report of C. gloeosporioides and C. karstii as causal agents of twig and shoot dieback disease in the Mediterranean region and the first report of C. gloeosporioides as a citrus pathogen in Albania

Asymptomatic congenital cystic adenomatoid malformation of the lung: Is it time to operate?

ObjectiveThe optimal management of congenital adenomatoid malformation of the lung remains controversial. Prenatal ultrasonographic analysis has increasingly discovered asymptomatic lesions, raising questions about the need for and timing of surgical treatment for asymptomatic congenital adenomatoid malformation. The aim of our study was to analyze the short-term postoperative outcome of symptomatic congenital adenomatoid malformations compared with asymptomatic malformations.MethodsAll the data of patients presenting with congenital adenomatoid malformations histologically diagnosed and operated on between 1998 and 2005 at our institution were retrospectively reviewed. Patients were divided into 2 groups: group A comprised asymptomatic infants, and group B comprised symptomatic infants. Major outcomes considered were the length of ventilation, pleural drainage, and hospital stay. Postoperative morbidity and mortality were also evaluated. Asymptomatic patients were further stratified for age at the time of the operation to evaluate whether age at surgical intervention affects the outcome. The Fisher's exact and Mann–Whitney tests were used as appropriate.ResultsFifty-seven patients were consecutively treated. Thirty-five patients were given diagnoses of asymptomatic lesions and were enrolled into group A, whereas 22 patients presenting with symptoms were entered into group B. The lengths of ventilation, pleural drainage, and hospital stay were significantly longer in patients with symptomatic congenital adenomatoid malformations. Moreover, symptomatic patients presented with a higher postoperative complication rate. The age-based stratification of asymptomatic children did not show any difference on either postoperative mortality or major outcome considered.ConclusionChildren with congenital adenomatoid malformations operated on when asymptomatic present a better short-term outcome than symptomatic children. In addition, age at the time of the operation does not negatively affect the outcome. Our findings support early surgical treatment for asymptomatic congenital adenomatoid malformation

Co-Infections by Fusarium circinatum and Phytophthora spp. on Pinus radiata: Complex Phenotypic and Molecular Interactions

This study investigated the complex phenotypic and genetic response of Monterey pine (Pinus radiata) seedlings to co-infections by F. circinatum, the causal agent of pine pitch canker disease, and the oomycetes Phytophthora xcambivora and P. parvispora. Monterey pine seedlings were wound-inoculated with each single pathogen and with the combinations F. circinatum/P. xcambivora and F. circinatum/P. parvispora. Initially, seedlings inoculated only with F. circinatum showed less severe symptoms than seedlings co-inoculated or inoculated only with P. xcambivora or P. parvispora. However, 30 days post-inoculation (dpi), all inoculated seedlings, including those inoculated only with F. circinatum, showed severe symptoms with no significant differences among treatments. The transcriptomic profiles of three genes encoding pathogenesis-related proteins, i.e., chitinase (PR3), thaumatin-like protein (PR5), phenylalanine ammonia-lyase (PAL), and the pyruvate decarboxylase (PDC)-encoding gene were analyzed at various time intervals after inoculation. In seedlings inoculated with single pathogens, F. circinatum stimulated the up-regulation of all genes, while between the two oomycetes, only P. xcambivora induced significant up-regulations. In seedlings co-inoculated with F. circinatum and P.xcambivora or P. parvispora none of the genes showed a significant over-expression 4 dpi. In contrast, at 11 dpi, significant up-regulation was observed for PR5 in the combination F. circinatum/P.xcambivora and PDC in the combination F. circinatum/P. parvispora, thus suggesting a possible synergism of multiple infections in triggering this plant defense mechanism

miRNA Signatures in Sera of Patients with Active Pulmonary Tuberculosis.

Several studies showed that assessing levels of specific circulating microRNAs (miRNAs) is a non-invasive, rapid, and accurate method for diagnosing diseases or detecting alterations in physiological conditions. We aimed to identify a serum miRNA signature to be used for the diagnosis of tuberculosis (TB). To account for variations due to the genetic makeup, we enrolled adults from two study settings in Europe and Africa. The following categories of subjects were considered: healthy (H), active pulmonary TB (PTB), active pulmonary TB, HIV co-infected (PTB/HIV), latent TB infection (LTBI), other pulmonary infections (OPI), and active extra-pulmonary TB (EPTB). Sera from 10 subjects of the same category were pooled and, after total RNA extraction, screened for miRNA levels by TaqMan low-density arrays. After identification of "relevant miRNAs", we refined the serum miRNA signature discriminating between H and PTB on individual subjects. Signatures were analyzed for their diagnostic performances using a multivariate logistic model and a Relevance Vector Machine (RVM) model. A leave-one-out-cross-validation (LOOCV) approach was adopted for assessing how both models could perform in practice. The analysis on pooled specimens identified selected miRNAs as discriminatory for the categories analyzed. On individual serum samples, we showed that 15 miRNAs serve as signature for H and PTB categories with a diagnostic accuracy of 82% (CI 70.2-90.0), and 77% (CI 64.2-85.9) in a RVM and a logistic classification model, respectively. Considering the different ethnicity, by selecting the specific signature for the European group (10 miRNAs) the diagnostic accuracy increased up to 83% (CI 68.1-92.1), and 81% (65.0-90.3), respectively. The African-specific signature (12 miRNAs) increased the diagnostic accuracy up to 95% (CI 76.4-99.1), and 100% (83.9-100.0), respectively. Serum miRNA signatures represent an interesting source of biomarkers for TB disease with the potential to discriminate between PTB and LTBI, but also among the other categories

First Report of Root and Basal Stem Rot Caused by Phytophthora cryptogea and P. inundata on Dwarf Banana in Italy

In Sicily (southern Italy) local cultivars of dwarf banana (Musa acuminata) are cultivated for edible fruit and as ornamental plants. During the summer of 2015, in an ornamental nursery of Aci San Filippo (Catania province), eastern Sicily, ten out of forty mature plants of dwarf banana grown in the field showed leaf chlorosis, wilt and sudden collapse of the entire plant associated with root and basal stem rot. Two Phytophthora species (overall 24 and 22 isolates, respectively) were consistently recovered directly from rotted roots and stems on BNPRA-HMI selective medium (Masago et al. 1977). Pure cultures of both species were obtained by single-hypha isolations. The first species formed slight petaloid colonies on potato dextrose agar (PDA) and slightly fluffy colonies on V-8 juice agar (V8A). It grew between 2 and 30°C, with an optimum of 25°C. On V8A discs flooded with non-sterile soil extract this species produced persistent, ovoid to obpyriform, non-papillate, internally proliferating sporangia (35 ..

Type D personality impairs Quality of Life, coping and short-term psychological outcome in patients attending an outpatient intensive program of cardiac rehabilitation

Background: Type D personality represents a risk factor for adverse outcome and impaired Quality of Life (QoL) in CHD patients. Only few studies investigated Type D patients following cardiac rehabilitation (CR). No study investigated Type D personality in Italian patients attending a CR program of 4 weeks. The aims of the study were a) to verify the presence of Type D personality among patients attending an Italian CR program; b) to investigate psychological health status, QoL and coping style of CR patients and c) to test the influence of Type D personality on CR patients outcome. Methods: Data from 59 patients attending an outpatient intensive program of 4 weeks of CR were collected at admission, and 1 month after discharge, using a set of self-report questionnaires. Variables were measured using CBAH, DS-14, Q-LES-Q and Brief COPE Scale. Results: The percentage of Type D personality found in the study sample was 39%. At admission Type D patients showed a significant lower level of psychological health status and QoL satisfaction compared to non Type D patients (p<0.05). After CR a significant percentage of Type D patients, despite an overall improvement, continued to show a clinically relevant psychological impairment in terms of anxiety (p=0.003), depressive mood (p=0.001), impairment in psychophysical well-being (p=0.002), perceived psychophysical stress (p=0.002), interpersonal difficulties (p<0.001), and social anxiety (p=0.045). Type D personality was also found to be associated with a significant greater use of maladaptive coping strategies (p<0.05). Conclusions: Type D personality played a significant clinically relevant role on psychological health outcome in CR. Type D personality patients reported a significant higher level of psychological impairment, in terms of anxiety, depressive mood, impairment in psychophysical wellbeing, perceived psychophysical stress, interpersonal difficulties, social anxiety, and a significant lower QoL, prior and after CR. Type D personality seemed also to be associated with maladaptive coping strategies. Importance of assessment for Type D personality is warranted in CR setting, as additional interventions seem required to enhance the outcome of these patients defined in letterature at high-risk

Potentialities of Usrp-Based Software Defined Radar Systems

International audienceSoftware Defined Radar is the latest trend in radar development. To handle enhanced radar signal processing techniques, advanced radars need to be able of generating various types of waveforms, such as frequency modulated or phase coded, and to perform multiple functions. The adoption of a Software Defined Radio system makes easier all these abilities. In this work, the implementation of a Software Defined Radar system for target tracking using the Universal Software Radio Peripheral platform is discussed. For the first time, an experimental characterization in terms of radar application is performed on the latest Universal Software Radio Peripheral NI2920, demonstrating a strongly improved target resolution with respect to the first generation platform