UNIVERSITA’ DEGLI STUDI DI UDINE in agreement with FONDAZIONE EDMUND MACH – Trento PhD School "Agricultural Science and Biotechnology" XXXI Cycle Coordinator: Prof. Giuseppe Firrao Doctoral Thesis Composition, dynamics and properties of apple and pear bark microbiota under different environmental conditions and disease management strategies Ph.D. Candidate Elena Arrigoni Supervisor Prof. Ilaria Pertot Tutor Dr. Michele Perazzolli DEFENCE YEAR 2019 Table of contents Abstract ................................................................................................................................ 1 Riassunto .............................................................................................................................. 4 CHAPTER I. Introduction ................................................................................................... 7 1.1 The plant microbiota ..................................................................................................... 7 1.2 Functions of the plant microbiota .................................................................................. 9 1.3 Economic relevance and history of apple ................................................................... 12 1.4 Apple scab ................................................................................................................... 14 1.5 Disease management of apple scab ............................................................................ 16 1.5.1 Chemical control of apple scab ............................................................................ 17 1.5.2 Cultural and biological control methods against apple scab ................................ 18 1.5.3 Scab-resistant apple cultivars ............................................................................... 19 1.6 Secondary diseases of apple ........................................................................................ 21 1.7 The study of microbial communities with sequencing technologies ........................... 23 1.7.1 The study of microbial diversity .......................................................................... 23 1.7.2 The Illumina sequencing technology.................................................................... 24 Aim of the thesis ................................................................................................................. 27 CHAPTER II. Tissue age and plant genotype affect the microbiota of apple and pear bark ............................................................................................................................................. 28 CHAPTER III. Environmental conditions and disease management strategies influence the composition of fungal and bacterial communities associated to apple barks ................ 65 CHAPTER IV. A fast and reliable method for Diplodia seriata inoculation of trunks and fungicide efficacy assessment on potted apple plants under greenhouse conditions .......... 89 CHAPTER V. Discussion ................................................................................................ 110 CHAPTER VI. Conclusions and future perspectives ...................................................... 114 References......................................................................................................................... 115 List of publications and training activities .................................................................... 133 Funding ............................................................................................................................. 136 Aknowledgements……………………………………………………………………..137 Abstract Plants host complex fungal and bacterial communities on and inside various organs. These microbial populations could have beneficial and detrimental effects on their host. Despite the importance of plant bark as potential reservoir of plant pathogens and beneficial microorganisms for perennial crops, no information is available on the composition of bark microbiota under different climatic and agronomic conditions. Due to its agricultural and economic relevance, apple (Malus domestica) was selected as model system, in order to characterise the composition and functional properties of the bark microbiota. Most of the commercially relevant apple cultivars are susceptible to several destructive diseases and require intense use of fungicides. Scab-resistant apple cultivars allow the reduction of fungicide treatments and are compatible with low-input disease management strategies. However, fungicides applied to control apple scab can also control secondary pathogens and the reduction of fungicide applications on scab-resistant cultivars may cause the outbreak of emerging pathogens hosted on bark tissues, as for example the canker agent Diplodia seriata. Although the risk of emerging secondary pathogens is real under low-input disease management strategies, no information is available on the composition and dynamics of bark-associated microbial communities. The main aims of this work are i) to develop metabarcoding-based approaches for the precise study of bark- associated microbial communities and to investigate proportions of plant pathogens and beneficial microorganisms associated with young (one year-old shoots) and old barks (three/four-year-old shoots), ii) to identify impacts of environmental factors (orchard location and climatic conditions), host properties (plant species, cultivar and tissue age) and agricultural practices (integrated and low-input disease management) on the bark microbiota composition and iii) to evaluate fungicide impacts against a scarcely studied canker agent of apple under controlled conditions. A metabarcoding approach was optimized to characterise the composition of bark- associated microbial communities of two apple and two pear cultivars (Chapter II). Several potential plant pathogenic fungi (e.g. Alternaria spp., Diplodia spp., Gibberella spp., Peltaster spp., Penicillium spp., Phoma spp., Rosellinia spp., Stemphylium spp. and Taphrina spp.) and bacteria (e.g. Pseudomonas spp., Clavibacter spp., Curtobacterium spp., Nocardia spp.and Pantoea spp.) were found in presence of beneficial fungi (e.g. Arthrinium spp., Aureobasidium spp., Cryptococcus spp., Rhodotorula spp., 1 Saccharomyces spp. and Sporobolomyces spp.) and bacteria (e.g. Bacillus spp., Brevibacillus spp., Burkholderia spp., Deinococcus spp., Lactobacillus spp., Methylobacterium spp. and Sphingomonas spp.). The composition of bark communities was affected by the tissue age, plant species and cultivar and it strongly differed between young (one year-old shoots) and old barks (three/four-year-old shoots). The optimised protocol was then applied to characterize the bark-associated communities of the scab- resistant apple cultivar Fujion under integrated and low-input disease management strategies in two different locations and consecutive seasons (Chapter III). In addition to the tissue age, orchard location and environmental conditions strongly affected fungal and bacterial richness and diversity of the bark communities. In particular, the orchard location affected the abundance of some potential plant pathogens, such as Alternaria spp., Cadophora spp., Diaporthe spp. Diplodia spp., Phoma spp. Curtobacterium spp., Pantoea spp. and Ralstonia spp. Moreover, the abundances of Cryptococcus, Leptosphaeria, Curtobacterium and Pseudomonas genera differed according to the disease management strategy, suggesting that it can shape the composition of plant-associated microbial communities. In particular, the low-input disease management strategy increased the abundances of sOTUs assigned to Alternaria, Cryptococcus, Rhodotorula, Curtobacterium, Methylobacterium, Rathayibacter and Rhizobacter, while the integrated disease management positively affected the abundances of Filobasidium, Erwinia and Pseudomonas. The functional characterization of apple secondary pathogens was then conducted on the canker agent D. seriata (Chapter IV). Diplodia seriata growth was inhibited by fungicides (captan, dithianon and fluazinam) in vitro although canker symptoms were not affected by dithianon on Fujion plants under greenhouse conditions. The overall work revealed a high complexity of the bark-associated microbial communities, highlighting bark as an overwintering site and reservoir of potential plant pathogens and beneficial microorganisms. Bark-associated fungal and bacterial communities were mainly affected by tissue age, orchard location, environmental conditions, disease management strategies, plant species and cultivars, indicating strong plasticity of the bark community to the environmental and host factors. However, cavities in the bark possibly protect microorganisms form perturbing factors and probably limit the efficacy of conventional fungicides against some secondary pathogens, such as D. seriata. Protocols optimised in this work can be further applied for the impact estimation of innovative agronomic practices (e.g. training systems and rain covers) on pathogenic and 2 beneficial communities and for the precise assessment of D. seriata incidence under field conditions. 3 Riassunto Le piante ospitano complesse comunità fungine e batteriche sulla superficie ed all’interno dei propri tessuti. Tali popolazioni microbiche possono esercitare funzioni benefiche o deleterie nei confronti dei propri ospiti. Nonostante l’importanza della corteccia come potenziale fonte di patogeni vegetali e microorganismi benefici per le colture perenni,