Recycling of waste compounds: chitosan of biological origin as a raw material for ENMs production

Chitosan is one of the most abundant biopolymers on earth, together with cellulose, and is easily obtainable from chitin-based waste (crustaceans, fungi, insects) using circular economy processes. Therefore, considerable interest is conferred to its application in developing new technologies for eco-sustainability in agriculture, especially in synthesizing nanomaterials. Furthermore, it is known that its beneficial properties, such as the induction of biological responses concerning plant defense against stresses, are enhanced when the polymer is in a nanometric form. Furthermore, chitosan nanostructures show better interaction with plant teguments and an appreciated durability and stability; hence, they are also suitable as carriers for bioactive molecules to be used as new-generation agronomic formulates for crop nutrition or protection. Given their potential in the future, this lesson will take an in-depth look at the properties and synthesis of chitosan nanoparticles, including some examples of plant pathology applications

Chitosan nanocarriers-mediated delivery of double-stranded RNA "in planta"

Agriculture is currently facing numerous challenges: the rapid rise of the world population, the consequent growth in food demand, the global decrease in crop yield. Particularly regarding the last issue, climate change is worsening the environmental stresses that commonly affect crops, and the use of resources – such as fertilizers and pesticides – is highly inefficient and pollutant. In this context, research is looking for new approaches to improve crop productivity by more efficient and environmentally friendly practices. It has been shown that nanomaterials are suitable for the development of cutting-edge technologies with the aim of improving the delivery of bioactive substances on plants and to promote their resistance to biotic and abiotic stresses. Among organic polymers, chitosan, if used in the nanoscale form, shows both properties; it can induce biological responses concerning plant defense against diseases and pathogen attack, and it is particularly suitable as a carrier for several molecules. Another innovative method for the defense of crops is the exploitation of the spray-induced gene silencing (SIGS) based on the activation of the so-called RNA-interference (RNAi). It involves exogenous double stranded RNAs (dsRNAs) targeting an essential pathogen gene, which trigger the RNAi pathway leading to the translational repression by degradation of target homologous mRNAs. In our case, the research aimed to verify the feasibility of dsRNA distribution on plant surface by means of functionalized chitosan nanoparticles (CH-NPs), thus allowing the protection of the doping agent and its efficient delivery. Here we show the preliminary results regarding the characterization of CH-NPs, their loading with dsRNAs and their interaction with the leaf surface of Nicotiana benthamiana plants. The effects of the dose-dependent distribution were analyzed by confocal microscopy upon incorporation of a fluorescent probe

Development of a non-chemical RNAi-based strategy for Amaranthus hybridus L. weed management

Weeds are one of the major issues in cropping systems, responsible for significant yield losses. Herbicide applications are the most effective strategy to control weeds, but stricter legislation has resulted in a significant reduction in the number of herbicides available on the market. Furthermore, the recent European legislation on the sustainable use of pesticides will require farmers to drastically reduce chemical use over the next ten years while promoting integrated weed management strategies that improve environmental sustainability and lower the risks to animal and human health. In addition, the over-reliance on chemical control has resulted in the evolution of resistant biotypes. As a result, new technologies to effectively manage weeds and weed resistance should be developed. In this regard, the development of a non-chemical weed control strategy based on RNA interference (RNAi) technology could: i) represent a potential non-chemical weed control strategy, ii) provide an emerging GMO-free strategy for managing invasive and resistant weeds, and iii) provide a valid opportunity to go inside the molecular mechanisms of weed biology. In this study, the acetolactate synthase (ALS) gene of Amaranthus hybridus L. has been used as the target to assess the effectiveness and applicability of in-vitro synthesized double-stranded RNAs (dsRNAs) direct application for endogenous gene silencing and weed control. A. hybridus is a monoecious and self-pollinated weed that has evolved multiple resistance to herbicides with different sites of action, including ALS inhibitors, which are the most used herbicides in soybean. ALS represents an ideal target for the development and future application of dsRNA-mediated gene silencing because it is an intronless, nucleotide-stable, and single-copy gene. We have produced dsRNAs of various lengths (ranging from 218 to 460bp) targeting three distinct ALS regions: the 5’- and 3’-ends, and a central region. dsRNAs molecules were transcribed in-vitro by T7 RNA polymerase and externally applied to the abaxial leaf surface of A. hybridus plants at 4-6 true leaves developmental stage by: i) mechanical inoculation, or ii) high-pressure spraying. Despite the expression of ALS gene transcripts was found to be lightly downregulated when synthetic 2 ALS-dsRNAs were applied, no phenotypic effects were observed. Our current research focuses on the determination of the effectiveness of ALS-dsRNAs silencing using agroinfiltration techniques, and on dsRNAs delivery techniques through the use of nanomaterials to maximize the effectiveness of gene silencing by exogenous dsRNAs application. This second approach was preliminary studied by RNA electrophoretic mobility of functionalized nanomaterial and by means of confocal microscopy on A. hybridus leaves. In parallel, we are examining the expression patterns of genes thought to be involved in the RNAi pathway in A. hybridus to verify if their expression is triggered by dsRNA applications

Comparison of leaf morpho-anatomical characters in Amaranthus spp.: phenotyping as an investigative tool for environmental and agricultural sciences

Plant phenotyping is an important tool that can provide insight into the interaction between plants and the environment, often as supporting information for genotype studies. The resulting knowledge can be useful in eco-physiological research, to understand how species adapt to their growing conditions and to biotic competition. In recent years, phenotyping techniques for the study of plant morpho-anatomical traits have developed in the field of the imaging analysis, starting from microscope images up to high scale acquisitions through remote sensing. In this work, we focused on the detailed study of single-leaf morphometric traits through the processing of photographic and confocal microscope acquisitions. Four species of Amaranthus were used, being plants of interest due to their high invasiveness into fields. Their morphological traits could become a useful tool to describe their adaptative responses and to define strategies for the sustainable management of the agro-ecosystem

Interactions between plant response to environment and fungal microbiome in developing maize silks in relation to mycotoxin risk

Maize (Zea mays L.) is one of the most important crops worldwide both in terms of yield and land surface used, which are constantly increasing. One of the most critical stages for maize reproduction and seed establishment is the emergence of silk from cob: silks are particularly susceptible to environmental stresses and represent a preferential entry route for mycotoxin-producing fungi such as Fusarium verticillioides and Aspergillus flavus (1). Moreover, from elongation to senescence, silks become a sink organ enriched in nutrients (e.g. Non-Structural Carbohydrates (NSC)), and a crossway for various primary and secondary metabolites. In maize silks, these metabolites are expected to be significantly affected by environmental stress conditions (2) and by the maturation stage of silk tissues themselves, possibly affecting the fungal colonization of the ear tissues. In temperate regions of cultivated maize, also pathogen growth and mycotoxin production are thought to be affected by environmental factors, such as alterations in temperature, rainfall and humidity (3), which are strictly related to climate change. The aim of this study is to investigate the impact of the environmental conditions on the fungal microbiome in maize developing silks at two different phenological stages. To do so, some eco-physiological parameters have been measured in 5 plots under contrasting climate conditions, and the complete fungal microbiome has been sequenced for each plot, both at the beginning of emergence and at the senescence of silks. In this study we expect to get new insights into the interplay of the environmental conditions, i.e. precipitation and temperature, and phenological stage of silks in determining the fungal microbiome of maize silks. We do believe that climate-induced plant response might be pivotal in shaping the microbiome communities by favouring some fungal groups and disfavouring others during early silk colonization. 1) Thompson, M. E. H. & Raizada M. N. Fungal pathogens of maize gaining free passage along the silk road. Pathogens, 7(4), 81 (2018). 2) Slewinski, T. L. Non-structural carbohydrate partitioning in grass stems: a target to increase yield stability, stress tolerance, and biofuel production. Journal Of Experimental Botany, Vol. 63, No. 13, pp. 4647-4670 (2012). 3) Magan, N. & Medina, A. Integrating gene expression, ecology and mycotoxin production by Fusarium and Aspergillus species in relation to interacting environmental factors. World Mycotoxin J. 9, 673–684 (2016)

Chitosan nanoparticles doped with dsRNA as a tool for sustainable viticulture: preliminary results

Agriculture is recently undergoing a period of transition towards sustainability, with the aim of providing sufficient food for the growing population by reducing the environmental impact. In this context, nanotechnologies are arousing interest in research thanks to the versatility and peculiar properties of some nanomaterials, which appear promising to make some agronomic practices, such as nutrition and crop protection, more eco-sustainable. Chitosan (CH) is an interesting organic polymer to be used to obtain nanoparticles (NPs), thanks to its biocompatibility and to the possibility of sourcing it through the circular economy. CH is known for its ability to induce several biological responses in plants concerning their growth and their defense against diseases, and it shows also good performances as a shuttle for a variety of molecules. This opens the possibility both to profit from the CH carrier function and its protective action against external agents, and to obtain a synergistic effect between it and the transported molecule. In our case, the goal is the functionalization of CH-NPs with specific dsRNA sequences of grapevine pathogens to exploit the RNA-interference (RNAi) mechanism, which has been suggested as an innovative strategy to limit pathogen infections. Preliminary results will be here presented, concerning the development of a protocol for the synthesis of CH-NPs, their characterization and the first information regarding their interaction with dsRNA sequences. Two variants of NPs have been produced (from chitosan as it is, or treated with hydrogen peroxide), which were doped with dsRNA sequences of Esca disease pathogens. The difference in synthesis procedures determined opposite interactions with nucleotides, resulting in a lower dimensional size and greater retention of the doping agent by the NPs obtained with untreated chitosan

Chitosan nanoparticles for sustainable agriculture: interactions with leaf surface and protective effect on dsRNA as functionalizing agent

Climate change and population growth are causing significant issues in the agricultural world, among which the worsening of environmental stresses suffered by crops and the inefficient use of resources must be highlighted. That’s why it’s necessary to find eco-sustainable solutions that can guarantee adequate production efficiency without affecting environmental health. Among the most advanced technologies, the development of nanomaterials partially replacing the conventional treatments with synthetic pesticides and fertilizers, stands out, given their more efficient transport of bioactive substances to plants and protection from damaging factors. Considering organic materials, nano-chitosan is even eco-compatible and obtainable through circular economy. Another innovation concerning the defense of crops is the exploitation of the so-called RNA-interference mechanism. Specific dsRNAs targeting an essential gene of a pathogen or weed can be applied exogenously, triggering a pathway that leads to gene silencing into the organism. A major issue, however, consists in the easy degradability of these sequences if applied naked, which makes the technology still unlikely for agricultural up-scaling. In this regard, the aim of our research was to verify the feasibility of dsRNA application on plants by means of functionalized chitosan nanoparticles (NPs), thus allowing its efficient delivery and protection. After defining the best synthesis protocol of NPs, these were used for different tests. To verify their ability to adhere to the leaf surface, they were observed by confocal microscope on two plant species thanks to a fluorescent probe. Subsequently, after their functionalization with nucleotides, their protective capacity was studied. These tests proved to be fundamental for the prosecution of the work concerning the evaluation of the formulation efficacy on plant pathogens