Abstract
The general introduction of this thesis, the importance of apple production worldwide and in South Tyrol, the main post-harvest diseases affecting apples, and the polyphasic approach to the study of phytopathogenic fungi are presented. Subsequently, in Chapter 1, the outcome of an investigation performed in packinghouses aiming to reveal the most important post-harvest diseases of stored apples was presented. The two-year investigation was performed in 2018 and 2019 in six cooperatives representing different cultivation areas in the Autonomous Province of Bozen-Bolzano (South Tyrol). The survey showed that the most common post-harvest pathogens of apple in this geographical area belong to the genera Penicillium, Botrytis, Alternaria, Diplodia, Neofabraea/Phlyctema, Colletotrichum and Fusarium. Prior to proceeding with further characterization of the different isolates obtained in this study, a single-spore isolation protocol was implemented by adapting the procedure of Choi et al. (1999). Chapter 2 addresses the characterization of Colletotrichum species based on multi-locus DNA sequences of four different genomic regions, as well as their morphological and pathogenicity assessment. A phylogenetic analysis based on multi-locus DNA sequences showed that the isolates obtained from apples with symptoms of bitter rot belonged to the species C. fioriniae, C. godetiae and C. salicis, which are part of the C. acutatum species complex. Moreover, the Colletotrichum isolates found in this study proved to be virulent on the cultivars 'Cripps Pink/Pink Lady®', 'Golden Delicious' and 'Roho 3615'/Evelina®. To the best of our knowledge, these three species referred to previously have so far never been reported as post-harvest pathogens of apple in Italy, even though the (re)-analysis of samples collected in the past indicates that C. godetiae and C. fioriniae have been occurring in Italy for at least a decade.
The species Cadophora luteo-olivacea is also categorized as a pathogen that induces latent post-harvest infections. This species causes side rot that is considered a minor post-harvest disease of apple. However, the symptoms caused by C. luteo-olivacea can be confused with those caused by Alternaria spp. or P. vagabunda, consequently only microbiological isolation on growth media and the molecular analysis by sequencing DNA marker segments can reliably distinguish these species. In Chapter 3, eleven fungal cultures, obtained from apples displaying post-harvest side rot symptoms, were identified as the genus Cadophora based on ITS (internal transcribed spacer) sequence data. For a more accurate taxonomic assignment, two additional molecular genetic loci,β-tubulin (TUB2) and translation elongation factor 1α (TEF-1α), of seven isolates obtained in this study were amplified and sequenced. A phylogenetic study including multi-locus sequence data of reference species of the genus finally allowed to determine all the isolates from South Tyrol as C. luteo-olivacea. A pathogenicity test performed on the cv. 'Golden Delicious' confirmed that the selected isolate was able to induce the disease. To the best of our knowledge, this is the first study reporting C. luteo-olivacea as a causal agent of post-harvest Side rot of domesticated apple in Italy.
Bull’s eye rot was the dominant latent post-harvest disease in the present study. In Chapter 4, 413 isolates, obtained during the two years of investigation, were identified as P. vagabunda, based on assessment of symptoms present on rotten apples, evaluation of morphological characteristics of fungal isolates, ITS barcoding of representative isolates, and multi-locus sequence typing based on three additional genomic regions (TUB2, TEF-1α, and mtSSU). In both investigation years, only six isolates were found that were identified as N. kienholzii. Finally, a pathogenicity test demonstrated different degrees of virulence among selected isolates of P. vagabunda and N. kienholzii on the cultivar ‘Golden Delicious’. To the best of our knowledge, this study is the first to report N. kienholzii as a post-harvest pathogen of apple in Italy, although our analyses suggest that the species may have been present in Italy for a longer time.
Numerous studies indicate that bull’s eye rot is one of the most frequent and damaging diseases occurring in stored apple fruits worldwide. However, studies about Neofabraea spp. biology are limited, mainly due to slow growth and scarce in vitro sporulation of these species. Considering that these species may differ biologically and require distinct management strategies in the field, their characterization through whole genome sequencing could represent a new resource useful for comparative genome analysis, which may also contribute to a better understanding of the evolution and taxonomy of this genus. Furthermore, this knowledge could be beneficial for functional genomics studies or the exploration of genetic elements governing vital biological and pathological processes. Therefore, in Chapter 5 the most suited sequencing strategy to obtain the whole genome sequence of main Neofabraea species was explored. Since the whole genomes of these species have never been sequenced, different fungal growth conditions, DNA extraction protocols, as well as next-generation sequencing (NGS) technologies such as Illumina MiSeq, Oxford Nanopore and PacBio SMRT Sequencing were evaluated.