Deciphering the role of plant microbiome on strawberry and raspberry growth, resistance, fruit quality and aroma

Besides their own adaptation strategies, plants might exploit microbial symbionts for overcoming both biotic and abiotic stresses and increase fitness. The current scenario of rapid climate change is demanding more sustainable agricultural management practices. The application of microbe-based products as a valid alternative to synthetic pesticides and fertilizers and their use to overcome stresses exacerbated by climate change, have been reviewed in the first part of this thesis. Berry fruits are widely cultivated and appreciated for their aromatic and nutraceutical properties. This thesis is focused on the role of plant and fruit microbiome on strawberry and raspberry growth, resistance, fruit quality and aroma. A taxonomical and functional description of the microbiome of different organs of three strawberry genotypes was performed both by traditional cultural dependent method and Next Generation Sequencing technique, highlighting a significant role of plant organs and genotype in determining the composition of microbial communities. Additionally, a selection of bacteria native of strawberry plants were isolated and screened for their plant growth promoting abilities and tested under the biotic stress of Xanthomonas fragariae infection and the abiotic stress of induced salinity. The monitoring of biometric parameters allowed the selection of a more restricted panel of bacterial strains, whose beneficial potential was tested in coordinated inoculations, or singularly. Raspberry plant was used for investigating the effect of cultivation method in determining fruit microbiome, and its consequent influence of berry quality and aroma. Interestingly, the cultivation method strongly influenced fruit nutraceutical traits, aroma and epiphytic bacterial biocoenosis. The involvement of the bacterial microbiota in fruit aroma determination was evaluated by performing GC–MS analysis of VOCs occurring in control, sterile and artificially reinoculated berries and by characterizing control and reinoculated berry microbiome. Differently treated berries showed significantly different aromatic profile, confirming the role of bacteria in fruit aroma development.

Organic fertilization of peach trees: implication on nitrogen availability, root growth and carbon distribution within plant

In the last years, sustainable horticulture has been increasing; however, to be successful this practice needs an efficient soil fertility management to maintain a high productivity and fruit quality standards. For this purpose composted organic materials from agri-food industry and municipal solid waste has been used as a source to replace chemical fertilizers and increase soil organic matter. To better understand the influence of compost application on soil fertility and plant growth, we carried out a study comparing organic and mineral nitrogen (N) fertilization in micro propagated plants, potted trees and commercial peach orchard with these aims: 1. evaluation of tree development, CO2 fixation and carbon partition to the different organs of two-years-old potted peach trees. 2. Determination of soil N concentration and nitrate-N effect on plant growth and root oxidative stress of micro propagated plant after increasing rates of N applications. 3. Assessment of soil chemical and biological fertility, tree growth and yield and fruit quality in a commercial orchard. The addition of compost at high rate was effective in increasing CO2 fixation, promoting root growth, shoot and fruit biomass. Furthermore, organic fertilizers influenced C partitioning, favoring C accumulation in roots, wood and fruits. The higher CO2 fixation was the result of a larger tree leaf area, rather than an increase in leaf photosynthetic efficiency, showing a stimulation of plant growth by application of compost. High concentrations of compost increased total soil N concentration, but were not effective in increasing nitrate-N soil concentration; in contrast mineral-N applications increased linearly soil nitrate-N, even at the lowest rate tested. Soil nitrate-N concentration influenced positively plant growth at low rate (60- 80 mg kg-1), whereas at high concentrations showed negative effects. In this trial, the decrease of root growth, as a response to excessive nitrate-N soil concentration, was not anticipated by root oxidative stress. Continuous annual applications of compost for 10 years enhanced soil organic matter content and total soil N concentration. Additionally, high rate of compost application (10 t ha-1 year-1) enhanced microbial biomass. On the other hand, different fertilizers management did not modify tree yield, but influenced fruit size and precocity index. The present data support the idea that organic fertilizers can be used successfully as a substitute of mineral fertilizers in fruit tree nutrient management, since they promote an increase of soil chemical and biological fertility, prevent excessive nitrate-N soil concentration, promote plant growth and potentially C sequestration into the soil.

Jasmonates and abscisic acid influence fruit ripening and plant water use: practical, physiological and morphological aspects

The aim of the present thesis was to better understand the physiological role of the phytohormones jasmonates (JAs) and abscisic acid (ABA) during fruit ripening in prospect of a possible field application of JAs and ABA to improve fruit yield and quality. In particular, the effects of exogenous application of these substances at different fruit developmental stages and under different experimental conditions were evaluated. Some aspects of the water relations upon ABA treatment were also analysed. Three fruit species, peach (Prunus persica L. Batsch), golden (Actinidia chinensis) and green kiwifruit (Actinidia deliciosa), and several of their cvs, were used for the trials. Different experimental models were adopted: fruits in planta, detached fruit, detached branches with fruit, girdled branches and micropropagated plants. The work was structured into four sets of experiments as follows: (i) Pre-harvest methyl jasmonate (MJ) application was performed at S3/S4 transition under field conditions in Redhaven peach; ethylene production, ripening index, fruit quality and shelf-life were assessed showing that MJ-treated fruit were firmer and thus less ripe than controls as confirmed by the Index of Absorbance Difference (IAD), but exhibited a shorter shelf-life due to an increase in ethylene production. Moreover, the time course of the expression of ethylene-, auxin- and other ripening-related genes was determined. Ripening-related ACO1 and ACS1 transcript accumulation was inhibited though transiently by MJ, and gene expression of the ethylene receptor ETR2 and of the ethylene-related transcription factor ERF2 was also altered. The time course of the expression of several auxin-related genes was strongly affected by MJ suggesting an increase in auxin biosynthesis, altered auxin conjugation and release as well as perception and transport; the need for a correct ethylene/auxin balance during ripening was confirmed. (ii) Pre- and post-harvest ABA applications were carried out under field conditions in Flaminia and O’Henry peach and Stark Red Gold nectarine fruit; ethylene production, ripening index, fruit quality and shelf-life were assessed. Results show that pre-harvest ABA applications increase fruit size and skin color intensity. Also post-harvest ABA treatments alter ripening-related parameters; in particular, while ethylene production is impaired in ABA-treated fruit soluble solids concentration (SSC) is enhanced. Following field ABA applications stem water potential was modified since ABA-treated peach trees retain more water. (iii) Pre- and post-harvest ABA and PDJ treatments were carried out in both kiwifruit species under field conditions at different fruit developmental stages and in post-harvest. Ripening index, fruit quality, plant transpiration, photosynthesis and stomatal conductance were assessed. Pre-harvest treatments enhance SSC in the two cvs and flesh color development in golden kiwifruit. Post-harvest applications of either ABA or ABA plus PDJ lead to increased SSC. In addition, ABA reduces gas exchanges in A. deliciosa. (iv) Spray, drench and dipping ABA treatments were performed in micropropagated peach plants and in peach and nectarine detached branches; plant water use and transpiration, biomass production and fruit dehydration were determined. In both plants and branches ABA significantly reduces water use and fruit dehydration. No negative effects on biomass production were detected. The present information, mainly arising from plant growth regulator application in a field environment, where plants have to cope with multiple biotic and abiotic stresses, may implement the perspectives for the use of these substances in the control of fruit ripening.

Soil application of Meliaceae derivatives: effect on carbon and nitrogen dynamics in the soil-plant system

The effect of soil incorporation of 7 Meliaceae derivatives (6 commercial neem cakes and leaves of Melia azedarach L.) on C and N dynamics and on nutrient availability to micropropagated GF677 rootstock was investigated. In a first laboratory incubation experiment the derivatives showed different N mineralization dynamics, generally well predicted by their C:N ratio and only partly by their initial N concentration. All derivatives increased microbial biomass C, thus representing a source of C for the soil microbial population. Soil addition of all neem cakes (8 g kg-1) and melia leaves (16 g kg-1) had a positive effect on plant growth and increased root N uptake and leaf green colour of micropropagated plants of GF677. In addition, the neem cakes characterized by higher nutrient concentration increased P and K concentration in shoot and leaves 68 days after the amendment. In another experiment, soil incorporation of 15N labeled melia leaves (16 g kg-1) had no effect on the total amount of plant N, however the percentage of melia derived-N of treated plants ranged between 0.8% and 34% during the experiment. At the end of the growing season, about 7% of N added as melia leaves was recovered in plant, while 70% of it was still present in soil. Real C mineralization and the priming effect induced by the addition of the derivatives were quantified by a natural 13C abundance method. The real C mineralization of the derivatives ranged between 22% and 40% of added-C. All the derivatives studied induced a positive priming effect and, 144 days after the amendment, the amount of C primed corresponded to 26% of added-C, for all the derivatives. Despite this substantial priming effect, the C balance of the soil, 144 days after the amendment, always resulted positive.

Development of guidelines for microbiological control in microbrewery

Over the past 15 years the Italian brewing scene showed interesting changes, especially with regard to the creation of many breweries with an annual production of less than 10,000 hectoliters. The beers produced by microbreweries are very susceptible to attack by spoilage micro-organisms that cause the deterioration of beer quality characteristics. In addition, most of the microbreweries do not practice heat treatments of stabilization and do not carry out quality checks on the product. The high presence of beer spoilage bacteria is an economic problem for the brewing industry because it can damage the brand and it causes high costs of product retrieval. This thesis project was aimed to study the management of the production process in the Italian microbreweries within a production less than 10,000 hl. In particular, the annual production, type of plant, yeast management, process management, cleaning and sanitizing of a representative sample of microbreweries were investigated. Furthermore was made a collection of samples in order to identify, with simple methods, what are spoilage bacteria more present in the Italian craft beers. 21% of the beers analysed were positive at the presence of lactic acid bacteria. These analytical data show the importance of understanding what are the weak points of the production process that cause the development of spoilage bacteria. Finally, the thesis examined the actual production of two microbreweries in order to understand the process management that can promote the growth of spoilage bacteria in beer and production plant. The analysis of the data for the two case studies was helpful to understand what are the critical points where the microorganisms are most frequently in contact with the product. The hygiene practices are crucial to ensure the quality of the finished product, especially in the case of non-pasteurized beer.

Sewage sludge as organic fertilizer in agriculture. A study of physico-chemical properties, agronomic efficiency and environmental safety, with a specific focus on tannery sludge

In recent decades, the use of organic fertilizers has gained increasing interest mainly for two reasons: their ability to improve soil fertility and the need to find a sustainable alternative to mineral and synthetic fertilizers. In this context, sewage sludge is a useful organic matrix that can be successfully used in agriculture, due to its chemical composition rich in organic matter, nitrogen, phosphorus and other micronutrients necessary for plant growth. This work investigated three indispensable aspects (i.e., physico-chemical properties, agronomic efficiency and environmental safety) of sewage sludge application as organic fertilizer, emphasizing the role of tannery sludge. In a comparison study with municipal sewage sludge, results showed that the targeted analyses applied (total carbon and nitrogen content, isotope ratio of carbon and nitrogen, infrared spectroscopy and thermal analysis) were able to discriminate tannery sludge from municipal ones, highlighting differences in composition due to the origin of the wastewater and the treatment processes used in the plants. Regarding agronomic efficiency, N bioavailability was tested in a selection of organic fertilizers, including tannery sludge and tannery sludge-based fertilizers. Specifically, the hot-water extractable N has proven to be a good chemical indicator, providing a rapid and reliable indication of N bioavailability in soil. Finally, the behavior of oxybenzone (an emerging organic contaminant detected in sewage sludge) in soils with different physico-chemical properties was studied. Through adsorption and desorption experiments, it was found that the mobility of oxybenzone is reduced in soils rich in organic matter. Furthermore, through spectroscopic methods (e.g., infrared spectroscopy and surface-enhanced Raman spectroscopy) the mechanisms of oxybenzone-humic acids interaction were studied, finding that H-bonds and π-π stacking were predominantly present.

Modelling, simulations and monitoring for sustainable plant and animal production systems through precision technique

Protected crop production is a modern and innovative approach to cultivating plants in a controlled environment to optimize growth, yield, and quality. This method involves using structures such as greenhouses or tunnels to create a sheltered environment. These productive solutions are characterized by a careful regulation of variables like temperature, humidity, light, and ventilation, which collectively contribute to creating an optimal microclimate for plant growth. Heating, cooling, and ventilation systems are used to maintain optimal conditions for plant growth, regardless of external weather fluctuations. Protected crop production plays a crucial role in addressing challenges posed by climate variability, population growth, and food security. Similarly, animal husbandry involves providing adequate nutrition, housing, medical care and environmental conditions to ensure animal welfare. Then, sustainability is a critical consideration in all forms of agriculture, including protected crop and animal production. Sustainability in animal production refers to the practice of producing animal products in a way that minimizes negative impacts on the environment, promotes animal welfare, and ensures the long-term viability of the industry. Then, the research activities performed during the PhD can be inserted exactly in the field of Precision Agriculture and Livestock farming. Here the focus is on the computational fluid dynamic (CFD) approach and environmental assessment applied to improve yield, resource efficiency, environmental sustainability, and cost savings. It represents a significant shift from traditional farming methods to a more technology-driven, data-driven, and environmentally conscious approach to crop and animal production. On one side, CFD is powerful and precise techniques of computer modeling and simulation of airflows and thermo-hygrometric parameters, that has been applied to optimize the growth environment of crops and the efficiency of ventilation in pig barns. On the other side, the sustainability aspect has been investigated and researched in terms of Life Cycle Assessment analyses.