“Flavescence dorée” (FD) is a grapevine quarantine disease associated with phytoplasmas and transmitted to healthy plants by insect vectors, mainly Scaphoideus titanus Ball. In Europe, FD was firstly reported in 1955, in France, and has dispersed through the central and southern European winegrowing regions, with a great impact in grapevine production. In Portugal, FD was identified for the first time in the “Vinhos Verdes” region (VVR) in 2006. Infected plants usually develop symptoms characterized by stunted growth, unriped cane wood, leaf rolling, yellowing or reddening (depending on berry colour), and shrivelled berries. Conventional management strategies rely mainly on the insecticide treatments, roguing infected plants and use of phytoplasma-free propagation material. However, these strategies are costly and could have undesirable environmental impacts. Development of sustainable approaches for FD control has been hampered by the lack of knowledge about phytoplasma biological properties and plant-pathogen interactions. The main aim of this thesis was to study the use of elicitors – methyl jasmonate, MeJA, salicylic acid, SA and benzothiadiazole, BTH – against phytoplasma-associated disease in grapevine. Elicitors have been successfully applied as preventive and environmentally friendly treatments for several diseases, but there is limited information on their effect on phytoplasmas. Since phytoplasmas are difficult to cultivate, Catharanthus roseus (commonly known as periwinkle) was used as a model system to study phytoplasma: host interactions as this plant species could present different degrees of symptom’ severity according with the phytoplasma strain. The specific goals of the present study were: (i) characterise FD phytoplasma strain in Vitis vinifera cv. Loureiro and study its effects on grapevine ultrastructure, growth, development, and productivity; (ii) understand the physiological and molecular responses triggered by the MeJA and SA application in FD-infected grapevines under field conditions; (iii) study the basal genomic and metabolomic profile of healthy and “aster yellows” phytoplasma-infected periwinkles and (iv) understand the role of MeJA and BTH in the induction of defence mechanisms against phytoplasmas. Field trials revealed that the phytoplasmas infecting grapevine cv. Loureiro belong to the 16SrV-D subgroup and show very low RFLP variability in the tuf and secY genes. Moreover, a two-year study demonstrated that FD infection delays grapevine development, leading to drastic production losses (up to 51%) and a decrease in berry quality, which may be partly linked to ultrastructural modifications, such as collapsed cells in the phloem cell, cell wall thickening and callose deposition, observed in the cells of infected plants. To understand grapevine responses to elicitors’ application – MeJA e SA – plant growth, productivity and berry quality parameters, and the expression of defence related genes were evaluated comparing healthy and FD infected plants treated with MeJA and SA. The application of 25 mM SA at flowering stage showed a partial reduction of the FD symptoms, since at veraison these plants did not show significant differences when compared to healthy plants for those parameters. However, this elicitor did not show significant effects in enhancing the productivity parameters. On the other hand, MeJA application in two subsequent years showed that the timing of application as well as the elicitor concentration are crucial to the possible management of this disease. In fact, 12.5 mM MeJA seemed to reduce the symptomatology in FD-infected grapevines, especially with the improvement of grapevine yield, also demonstrating a tendency to improve plant growth, development, and productivity parameters. On the other hand, 25 mM MeJA triggered an upregulation of Thau I, Thau II, Osm and PAL (in 2015) and Protα5s, CHIT4c, PIN, PGIP and GLU (in 2016) genes. MeJA treatments also significantly increased saponin and proline synthesis in FD-infected plants. To assess the impact of phytoplasmas on plant metabolism, three clones of micropropagated periwinkles were used: healthy and aster yellows infected shoots, showing mild (strain AY107) and severe (strain Hyd8) symptomatology degrees, respectively. In those trials it was possible to analyse the metabolomic profile of phytoplasma-infected periwinkles and the induction of defence mechanisms against these pathogens after the elicitation with MeJA and BTH. To that end samples were collected 1 and 4 days after elicitation (dae) treated with 12.5 or 25 mM MeJA and 3.5 or 7 mM BTH. The concentrations of 25 mM MeJA and 3.5 mM BTH showed the lowest malondialdehyde production four dae in infected shoots and an increase by 29% in flavonoid content in AY107 infected shoots one dae. This study also revealed that untreatedinfected shoots of AY107 strain presented higher content of ABA, which could be an indication of active phytoplasma infection as well as both elicitors reduced the phytoplasma symptomatology decreasing the ABA content. In the Hyd8 infected shoots 7 mM BTH and 12.5 mM MeJA resulted the most efficient at 1 and 4 dae, respectively, at biochemistry and molecular levels. Finally, a basal metabolomic analysis of the aforementioned periwinkle strain shoots identified 13 alkaloids and 11 phenolic compounds and showed the expression of genes related to alkaloid (AS, STR and PRX) and phenylpropanoid (PAL and CHS) biosynthesis pathways. Upregulation of pal, chs, as and str genes only in plants infected by both “aster yellows” strains suggested that the phytoplasma infection influences both alkaloid and phenolic biosynthetic pathways at a metabolic and molecular levels. Overall, the present PhD thesis put lights on the role of the studied elicitors as a novel and promising approach to manage phytoplasma diseases, developing and implementing new green practices towards a more sustainable and/or organic viticulture.
| Date of Award | 6 Dec 2021 |
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| Original language | English |
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| Awarding Institution | - Universidade Católica Portuguesa
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| Supervisor | Susana Maria Pinto de Carvalho (Supervisor), Marta Vasconcelos (Co-Supervisor) & Assunta Bertaccini (Co-Supervisor) |
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