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.