Microbiological and biochemical investigations on the aetiology of apple replant disease

Soil health is a key factor for the conservation of biodiverse ecosystems and sustainable agricultural production. Unfortunately, land exploitation due to intense monoculture tends to deplete and exhaust soil resources, giving rise to cultivation problems and harvest reduction. An example is apple replant disease (ARD), one of the major problems of apple production, occurring when apple trees are subsequently planted in the same soil. Despite the efforts in research, the exact aetiology of this disease is still uncertain. The present work investigates the microbial and biochemical complexity of agricultural soils using next generation technologies and especially focusing on the clearing the causes of apple replant disease. In Chapters II and III, the attention is focused on the microbial communities in ARD-affected soils in field and greenhouse experiments, using next generation sequencing (NGS). In the first situation, soil bacterial and fungal communities were compared in fumigated (dazomet 99%) and untreated soils in an apple orchard where fumigation relived ARD symptoms on apple trees. In Chapter III, the three different hypothesis on the onset of ARD (complex of microbial pathogens – presence of toxins released by old apple trees – nutrient imbalances in soil) were tested by the application of different treatments on ARD-affected soil planted with M9 rootstock in the greenhouse. The results show a change in the microbial balance in ARD-affected soils, with an increased presence in beneficial microorganisms in healthy soils compared to higher concentration of potential pathogens in sick soils. There was also an indication that toxins released by old apple trees might have a role in influencing negatively plant growth. In Chapter IV, a comprehensive meta-analysis of all the available ARD soil microbial sequencing studies was performed, in order to assemble the vastest data set and analyse it with the same bioinformatics tools to individuate the main drivers in ARD-affected soils. This meta-study confirms the difference in soil microbial communities in ARD affected soils, where a complex of phytopathogenic and nematophagous microorganisms was found, but highlighted also a change in microbial associations, that could be caused by a change in soil chemistry or metabolome. Therefore, more attention should be directed to the measurement of soil parameters, since this would help classify ARD as opportunistic microbial infectious disease, which could be shaped by a complex combination of environmental parameters affecting microbial communities, ultimately culminating in plant disease. In Chapter V, the changes in soil phenolic profile after the addition of apple roots were analysed using targeted metabolomics, since autotoxins produced by old apple trees were among the hypothesized causes of ARD. The autotoxicity of old apple roots was also measured on apple seedlings. The addition of apple roots damaged the seedlings, and, at the same time, a high concentration of phlorizin, a phenolic compound known to be phytotoxic, was assessed. The presence of this substance, right after the explanting of apple trees, could contribute to build up the necessary conditions for the onset of ARD. Finally, in Chapter VI, the attention moves to vineyards, another cultivation that highly exploits the soil, and where new more environmentally compatible ways of managing plants are emerging. In this work, soil microbial communities in vineyards managed with organic, biodynamic and biodynamic with green manure methods were analyzed with NGS. The green manure was the main input of soil microbial biodiversity, with a higher abundance of microorganisms involved in the nitrogen cycle and in the degradation of organic matter. The results of this work provide insights in the microbial and biochemical complexity of agricultural soils in apple orchards and vineyards, with the ultimate scope of understanding better the multiple mechanisms that rule them, to develop a more environmentally sound management that would improve harvest without long-lasting negative consequences on the ecosystems.