How to counteract the Ailanthus altissima invasion: could Verticillium have a role in the biological control of the ‘‘Tree of Heaven’’?

Invasive species are one of the leading drivers of global environmental change because of their detrimental impacts at both ecological and economic levels. Preventing the colonization or establishment of IAPS, let alone suppressing or eradicating them, has posed a major challenge to nature conservation for decades. Most research and nature conservation measures focus on the effectiveness of mechanical or chemical removal and control of IAPS on invaded sites, so knowledge on the effectiveness of these restoration measures is widely available. Among the IAPS control, the management of Ailanthus is particularly difficult due the ineffectiveness of the common mechanical practices and the limitation of chemicals distribution. In this thesis, the Verticillium wilt disease affecting the Tree of Heaven was investigated at different levels to evaluate the potential use of the fungal pathogen causing the disease as a biological, green and sustainable method for the control of this IAPS. The first case of Ailanthus decay in Tuscany was found in 2016 and was attributed to a specific causal agent identified morphologically and molecularly as Verticillium dahliae strain VdGL16 (ITS sequence has been deposited in GenBank with the accession number MK474459). Then, although Verticillium species are generally known to be polyphagous, host-range tests, consisting in artificial inoculations of more than 40 species (woody and herbaceous plants) with liquid cultures of V. dahliae strain VdGL16, revealed that isolated fungus has a restricted host range, severely affecting only ten species of five botanical families (Asteraceae, Lamiaceae, Leguminoseae, Linaceae and Solanaceae). This work also confirmed the high susceptibility of Ailanthus to Verticillium strain VdGL16 after artificial inoculations, independently to the source (seedlings or trees). In addition, results from intraspecific resistance screening of nine seed sources across Italy revealed that Ailanthus provenances from all the six sampled regions were susceptible. Stem inoculated adult plants exhibited abundant production of epicormic sprouts along the stem within six months, and most of these sprouts wilted following initial dieback of the main stem; sprouting from the crown was also intense. Petioles and rachises tissues of leaves fallen from infected trees were a good source for re-isolation of the pathogen and they could effectively transfer the fungus to healthyindividuals, representing on one side a potential risk for non-target effects, to the other hand a secondary vector for Ailanthus infections via roots. The Verticillium wilt disease was deeply investigated in the model species Solanum melongena cv. Violetta di Rimini (previously resulted susceptible to V. dahliae strain VdGL16), from the time of inoculation to 25 dpi, elucidating ecophysiological and biochemical responses in leaves at different ages (mature, intermediate and young; ML, IL and YL). This activity revealed that the disease developed rapidly and severely, as confirmed by the visible leaf injuries that firstly occurred on older leaves and then on younger ones, followed by a relentless early leaf senescence and plant death. Photosynthetic apparatus was significantly damaged by infection, as confirmed by the marked reduction of photosynthetic rate associated with stomatal (reduced stomatal conductance) and mesophyll (concomitant increase of intercellular CO2 concentration) limitations, that occurred from 8 wpi in both, ML and IL. Cell membrane integrity was compromised, and phylloptosis/death started to occurr after 8 dpi in ML, and later in IL. Indeed, in IL, at 18 dpi, the consumption of secondary metabolites suggested that antioxidant- and antimicrobial-defence responses were activated. However, photoinhibition, oxidative stress and water deficit were observed at the end of the experiment. These mechanisms were observed also in YL (generally, less affected by older oners) as confirmed by the strong increase of tannins followed by accumulation of other phenylpropanoids. Despite plant growth was maintained, reduction of leaf area and water deficit occurred. Overall, this study highlighted the capacity of eggplant to activate dynamic biochemical mechanisms in response to fungal infection, even in susceptible genotypes, representing a starting point for comparisons with other resistant material for selection. Importantly, this study allowed to develop an investigation protocol to be applied to the more complicated Ailanthus-Verticillium patho-system. Thus, a ten-week open air pot experiment was then set to investigate the ecophysiological and biochemical responses of Ailanthus trees artificially inoculated in the trunk with the V. dahliae strain VdGL16. Here, inoculated plants showed visible injuries starting from two wpi, that progressively developed until a final severe defoliation. The fungal infection rapidly compromised the plant water status, and photosynthesis was impaired due to both stomatal and mesophyll limitations from 4 wpi, with subsequent detrimental effects also on PSII activity. Moreover, the disease altered the nutrient translocation, as confirmed by cation and carbohydrate contents, probably due to a consumption of simple sugars and starch reserves without replacement of new photosynthesized. An accumulation of osmolytes (abscisic acid and proline) and phenylalanine (a precursor of phenylpropanoids) was also reported at 8 wpi, this being a response mechanism that needs to be further elucidated. However, the activation delay of such defence strategy inevitably did not avoid the premature defoliation of plants. Overall, this work, performed in partially controlled conditions, confirmed Verticillium as highly virulent against Ailanthus, further highlighting the huge potential of the pathogen for an efficient control of the invasive species. Furthermore, this work represented a fundamental step for moving to a field experiment where the same approach was adopted in an urban area. The field experiment was performed in an urban site characterized by the presence of monumental walls of Pisa old town, chosen because representative of other scenarios in which the management of Ailanthus (and other invasive plant species) commonly results particularly difficult. In this work, four selected Ailanthus trees were inoculated in summer 2017 using a liquid culture of V. dahliae strain VDGL16 using a stem injection at breast height of the trunk. First symptoms occurred in late spring of 2018 and became more evident in 2019, with a gradual phylloptosis and reduction in flowering. Verticillium was successfully re-isolated from inoculated plants. In 2020, inoculated Ailanthus trees showed a severe defoliation and visible damages at wood level. Regarding dendrochronological analyses, infection generally induced variations especially in vessel area and density, and these alterations, even if not activated at the same time, could represent a response of infected plants to compensate the presence of several vessel occlusions and avoid the risk of secondary issues such as embolism or cavitation. Anatomical observations of the wood of transversal sections collected at 1 and 4 m high transversal sections revealed vessel occlusions due to (i) mycelium, (ii) tyloses and (iii) other structures. Specifically, in 2018 the presence of tyloses could be considered an attempt to limit fungal colonization by creating physical barriers. However, the tyloses were not able to arrest the spread of the pathogen through the plant, which finally died. Indeed, by occluding many vessels, the presence of mycelium aggravated the symptoms of infected Ailanthus trees. This urban trial allowed to validate the method previously tested only in protected facilities, confirming the effectiveness of V. dahliae strain VDGL16 inoculum. Even considering all potential risk factors, among which it is necessary to also include leaves fallen from infected trees as potential disease vectors to non-target hosts, the V. dahliae strain VDGL16 inoculation appears suitable, especially in monumental and urban areas where Ailanthus is difficult to manage and the presence of other non-target hosts is rare. However, in the optic of an effective distribution of a Verticillium-based products, it seemed necessary to characterize the Ailanthus populations spread all over Italy, as well as in other European areas. So a molecular study on 129 Ailanthus leaf samples collected from both Italian and European locations is reported. Here, the analyses revealed that the collected genotypes are differentiated and there is no relationship among provenances and genetic groupings. This could mean that a very high biodiversity exists in the species, probably due to the easy dispersal of seeds mediated by different vectors, as well as the rapid spread and growth of the species. The high adaptability of Ailanthus to several environmental conditions opens a further research question about the potential role of Ailanthus provenance in the grade of susceptibility of this species to the biological control agent , a gap that will be addressed with further investigations that will be carried out in both controlled and field conditions