Development of SSR markers and genetic diversity analysis in enset (Ensete ventricosum (Welw.) Cheesman), an orphan food security crop from Southern Ethiopia

Enset (Ensete ventricosum (Welw.) Cheesman; Musaceae) is a multipurpose drought-tolerant food security crop with high conservation and improvement concern in Ethiopia, where it supplements the human calorie requirements of around 20 million people. The crop also has an enormous potential in other regions of Sub-Saharan Africa, where it is known only as a wild plant. Despite its potential, genetic and genomic studies supporting breeding programs and conservation efforts are very limited. Molecular methods would substantially improve current conventional approaches. Here we report the development of the first set of SSR markers from enset, their cross-transferability to Musa spp., and their application in genetic diversity, relationship and structure assessments in wild and cultivated enset germplasm

Indigenous knowledge, use and on-farm management of enset (Ensete ventricosum (Welw.) Cheesman) diversity in Wolaita, Southern Ethiopia

Background Ensete ventricosum(Welw.) Cheesman is a major food security crop in Southern Ethiopia, where it was originally domesticated and during millennia became pivotal crop around which an entire farming system has developed. Although its cultivation is highly localized, the enset-based farming system provides sustenance to more than 20 million people. Precise ethnobotanical information of intra-specific enset diversity and local knowledge on how communities maintain, manage and benefit from enset genetic resources is imperative for the promotion, conservation and improvement of this crop and its farming system. Methods This study was conducted in Southern Ethiopia among the Wolaita 'enset culture' community. The research sample consisted of 270 households from 12 Kebeles(villages) representing three agro-ecological ranges. By establishing Participatory Rural Appraisal (PRA) based interactions and applying ethnobotanical interviewing methods of free-listing and open-ended questionnaires, information on the use and management of enset diversity, and its associated folk-biosystematics, food traditions and material culture was collected and analyzed. Results While enset agriculture is seen as cultural heritage and identity for the Wolaita, enset intra-specific diversity holds scenic, prestige and symbolic values for the household. In the present study we recorded 67 enset landraces under cultivation, and through a comprehensive literature review we identified 28 landraces reported from other areas of Wolaita, but not encountered in our survey. Landraces, identified using 11 descriptors primarily related to agro-morphological traits, are named after perceived places of origin, agro-morphological characteristics and cooking quality attributes. Folk classification of enset is based on its domestication status, 'gender', agro-ecological adaptability and landrace suitability for different food and other uses (fiber, feed, medicinal). Enset as a food crop is used to prepare 10 different dishes in Wolaita, 8 of which are exclusively prepared using enset, and their consumption ranges from daily staple to specialty food in festive occasions and ceremonies. On-farm landrace diversity and richness is guided by household needs; its dynamics is managed through regular propagation, harvesting restrain, control of landrace composition and arrangement in the enset homegardens. Conclusions This study reported on the knowledge system, socio-cultural process and community practices that drive the maintenance of intra-specific on-farm enset diversity in Wolaita, Southern Ethiopia. The information is crucial for developing community based complementaryin situ andex situconservation strategies to foster conservation of enset genetic resources and associated indigenous knowledge system

Nitrogen Fertilizers Shape the Composition and Predicted Functions of the Microbiota of Field-Grown Tomato Plants

The microbial communities thriving at the root_soil interface have the potential to improve plant growth and sustainable crop production. Yet, how agricultural practices, such as the application of either mineral or organic nitrogen fertilizers, impact on the composition and functions of these communities remains to be fully elucidated. By deploying a two-pronged 16S rRNA gene sequencing and predictive metagenomics approach, we demonstrated that the bacterial microbiota of field-grown tomato (Solanum lycopersicum) plants is the product of a selective process that progressively differentiates between rhizosphere and root microhabitats. This process initiates as early as plants are in a nursery stage and it is then more marked at late developmental stages, in particular at harvest. This selection acts on both the bacterial relative abundances and phylogenetic assignments, with a bias for the enrichment of members of the phylum Actinobacteria in the root compartment. Digestate-based and mineral-based nitrogen fertilizers trigger a distinct bacterial enrichment in both rhizosphere and root microhabitats. This compositional diversification mirrors a predicted functional diversification of the root-inhabiting communities, manifested predominantly by the differential enrichment of genes associated to ABC transporters and the two-component system. Together, our data suggest that the microbiota thriving at the tomato root_soil interface is modulated by and in responses to the type of nitrogen fertilizer applied to the field

Plant antibodies for human antifungal therapy

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting β-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases

Recombinant antibodies for uses in agro-food diagnostics

Le micotossine sono metaboliti secondari prodotti da diversi funghi che appartengono principalmente a tre generi molto diffusi: Aspergillus, Penicillium e Fusarium. In particolare, l’aflatossina B1 è un contaminante molto comune che si può ritrovare in un’ampia gamma di prodotti alimentari. L’attenzione rivolta verso questi contaminanti è giustificata dai gravi effetti (teratogeni, cancerogeni, estrogeni, neurotossici e di immunosoppressione) sulla salute dell’uomo e degli animali conseguenti all’assunzione attraverso il cibo. Una contaminazione da micotossine può avvenire in campo prima e/o durante il raccolto oppure nella successiva fase di conservazione e processamento delle derrate alimentari. Sebbene l’uso di fitofarmaci e buone pratiche colturali possano ridurre il rischio di contaminazioni, la completa eliminazione delle micotossine dai prodotti alimentari non è ad oggi realisticamente raggiungibile. Pertanto la diagnostica rimane uno strumento fondamentale per ridurre il rischio correlato all’assunzione di cibi contaminati. Scopo di questo lavoro è stato l’isolamento e la caratterizzazione di nuovi immuno-reagenti per la diagnostica agro-alimentare, ed in particolare anticorpi ricombinanti da esprimere in sistemi a basso costo alternativi al sistema classico di colture di cellule di mammifero. Inizialmente si è tentato di isolare anticorpi, diretti al riconoscimento dell’aflatossina B1 e dell’ocratossina A, da due differenti repertori sintetici di anticorpi ad esposizione su fago (F8 ed ETH-2-Gold). E’ stata scelta questa metodologia di selezione in quanto risulta semplice, poco costosa ed ha consentito di operare la selezione su più antigeni contemporaneamente. Purtroppo da entrambi i repertori utilizzati non sono stati isolati anticorpi specifici per le tossine d’interesse. Pertanto, per isolare anticorpi specifici almeno per l’aflatossina B1, si è deciso di utilizzare il sistema classico di selezione da ibridomi murini. Topi Balb/c sono stati immunizzati con l’aflatossina B1 coniugata alla Keyhole Limpet Hemocyanin (KLH) come carrier proteico, questo per ovviare alla scarsa immunogenicità della tossina dovuta alle sue ridotte dimensioni (~300 Da). Dagli ibridomi ottenuti sono stati isolati tre differenti cloni cellulari secernenti anticorpi monoclonali in grado di riconoscere l’aflatossina B1 libera in soluzione in ELISA competitivo. I geni codificanti le catene pesanti e leggere degli anticorpi sono stati amplificati dal cDNA dei tre ibridomi utilizzando oligonucleotidi degenerati (descritti in letteratura per la catena gamma - Wang et al., 2000 Journal of Immunological Methods 233: 167-177) e disegnando nuovi oligonucleotidi basati su sequenze note per la catena leggera lambda (Kabat et al., 1991; Sequences of Proteins of Immunological Interest, 5th edit., US Department of Health and Human Services, US Government Printing Office). I geni delle catene leggere e pesanti sono stati quindi separatamente clonati in un vettore per l’espressione transiente in pianta mediata da A. tumefaciens (pBI) e piante di N. benthamiana sono state co-agroinfiltrate con entrambi i costrutti per ciascun clone (2D2_G1 e 9E11_D5). L’analisi western blot degli estratti di pianta ha mostrato alti livelli di espressione degli anticorpi, mentre il corretto assemblaggio e la funzionalità delle IgG murine è stata valutata mediante ELISA. Gli anticorpi, purificati dalle foglie infiltrate, hanno dimostrato di riconoscere in ELISA l’aflatossina B1 con la medesima specificità ed affinità dei rispettivi anticorpi purificati da terreno di crescita degli ibridomi murini. Inoltre, le sequenze delle regioni variabili dell’anticorpo monoclonale 2D2_G1, sono state ingegnerizzate per ottenere due differenti formati ricombinanti dell’anticorpo: il scFv e il scFv-Fc. Il scFv(2D2_G1) è stato espresso in E. coli e dopo la purificazione la specificità dell’anticorpo per l’aflatossina B1 è stata valutata per ELISA ed analisi SPR (Surface Plasmon Resonance). In parallelo, il formato scFv-Fc è stato clonato in un vettore per l’espressione transiente in pianta mediata da A. tumefaciens (pBI) ed infiltrato in piante di N. benthamiana. L’analisi western blot degli estratti di pianta e l’ELISA hanno confermato il corretto assemblaggio e la funzionalità di questo formato ricombinante dell’anticorpo. In conclusione, sono stati isolati due differenti anticorpi monoclonali murini diretti al riconoscimento dell’aflatossina B1 ed i geni codificanti per gli anticorpi sono stati clonati ed ingegnerizzati. Tutti i formati anticorpali ricombinanti espressi in sistemi eterologhi hanno mantenuto la specificità per l’antigene dei mAb murini, rinforzando così l’idea delle piante come bioreattori efficienti ed economici per la produzione di IgG in alternativa ai sistemi classici di colture di cellule di mammifero, ed aprendo nuovi orizzonti applicativi nell’ambito della diagnostica agro-alimentare.Mycotoxins are secondary metabolites produced by several fungi belonging mainly to the genera: Aspergillus, Penicillium and Fusarium. In particular, aflatoxin B1 is a common contaminant occuring in a wide range of important raw food commodities. Consumption of mycotoxin-contaminated food or feed eventually leads to teratogenic, cancerogenic, oestrogenic, neurotoxic, and immunosuppresive effects in humans and/or animals. Mycotoxin contamination may occur in the field before and/or during harvesting, or in the storage and processing. Although the use of pesticides and good agronomic practices may reduce mycotoxin accumulation, eradication is still a major challenge. Therefore, diagnostics remains a fundamental tool to reduce risks associated to the assumption of contaminated food. To this aim, we concentrated our efforts on the isolation and characterization of new diagnostic immuno-reagents such as recombinant antibodies to be expressed in cost-effective systems as an alternative to classical mammalian cell culture system. Initially, we attempted to isolate antibodies against aflatoxin B1 and ochratoxin A from two different synthetic antibody phage display libraries (F8 and ETH-2-Gold). In principle, this approach could represent a straightforward solution, as the methodology is simple, inexpensive, enabling simultaneous processing of several antigens. Unfortunatly, no antibody specific to these toxins were isolated from both libraries.Therefore, to isolate antibodies at least against aflatoxin B1, Balb/c mice were immunized with the toxin conjugated to the carrier Keyhole Limpet Hemocyanin (KLH), due to the poorly immunogenic size of this molecule (~300 Da). Three different clones were isolated from hybridomas and all derived monoclonal antibodies were able to recognize free aflatoxin B1 in competitive ELISA. The genes encoding the antibody heavy and light chains were amplified from the cDNA of the three hybridomas using a set of degenerated primers (as described in literature for the gamma chain - Wang et al., 2000 Journal of Immunological Methods 233: 167-177) and designing Kabat sequence-based primers for the lambda chain (Kabat et al., 1991; Sequences of Proteins of Immunological Interest, 5th edit., US Department of Health and Human Services, US Government Printing Office). Light and heavy chain genes were separately cloned in an Agrobacterium-mediated plant expression vector (pBI) and N. benthamiana plants were co-agroinfiltrated with both constructs for each clone (2D2_G1 and 9E11_D5). Western blot analysis of plant extracts revealed high expression levels of antibody chains, while the correct assembly and functionality of murine IgGs was evaluated by ELISA. The antibodies, purified from infiltrated leaves, bind aflatoxin B1 to the same extent as the cognate antibodies purified from murine hybridomas in ELISA. Moreover, starting from the variable regions sequences of mAb 2D2_G1, two different recombinant formats were engineered: the scFv and the scFv-Fc. The scFv(2D2_G1) was expressed in E. coli and after purification, antibody specificity to aflatoxin B1 was evaluated by ELISA and SPR (Surface Plasmon Resonance) analysis. In parallel, the scFv-Fc format was cloned in an Agrobacterium-mediated plant expression vector (pBI) and N. benthamiana plants infiltrated. Western blot analysis of plant extracts and ELISA confirmed the correct assembly and functionality of this recombinant antibody format. In conclusion, two different murine monoclonal antibodies against aflatoxin B1 have been generated and corresponding antibody genes cloned and engineered. All recombinant antibody formats expressed in heterologous systems retain the specificity of the original mAbs reinforcing the idea that plants are convenient bioreactors for the production of IgG alternative to classical mammalian cell culture systems, thus opening new horizons for both agricultural and diagnostic applications.Dottorato di ricerca in Biotecnologie vegetal

Design of a Diagnostic Immunoassay for Aflatoxin M1 Based on a Plant-Produced Antibody

A new green competitive ELISA for aflatoxin M1 quantification in raw milk was developed. This diagnostic tool is based on an anti AFM1 mAb produced by plant molecular farming in alternative to classical systems. Our assay, showing an IC50 below 25 ng/L, fits with the requirements of EU legislation limits for AFM1 (50 ng/L). Optimal accuracy was achieved in correspondence of the decision levels (25 and 50 ng/L), and the assay enabled AFM1 quantification in the range 5–110 ng/L, with limit of detection 3 ng/L. Moreover, to evaluate a real applicability in diagnostics, raw milk-spiked samples were analysed, achieving satisfactory recovery rates of AFM1. In conclusion, an efficient and ready-to-use diagnostic assay for the quantification of aflatoxin M1 in milk, based on a plant-produced recombinant mAb, has been successfully developed

Towards Predictive Modeling of Sorghum Biomass Yields Using Fraction of Absorbed Photosynthetically Active Radiation Derived from Sentinel-2 Satellite Imagery and Supervised Machine Learning Techniques

Sorghum crop is grown under tropical and temperate latitudes for several purposes including production of health promoting food from the kernel and forage and biofuels from aboveground biomass. One of the concerns of policy-makers and sorghum growers is to cost-effectively predict biomass yields early during the cropping season to improve biomass and biofuel management. The objective of this study was to investigate if Sentinel-2 satellite images could be used to predict within-season biomass sorghum yields in the Mediterranean region. Thirteen machine learning algorithms were tested on fortnightly Sentinel-2A and Sentinel-2B estimates of the fraction of Absorbed Photosynthetically Active Radiation (fAPAR) in combination with in situ aboveground biomass yields from demonstrative fields in Italy. A gradient boosting algorithm implementing the xgbtree method was the best predictive model as it was satisfactorily implemented anywhere from May to July. The best prediction time was the month of May followed by May–June and May–July. To the best of our knowledge, this work represents the first time Sentinel-2-derived fAPAR is used in sorghum biomass predictive modeling. The results from this study will help farmers improve their sorghum biomass business operations and policy-makers and extension services improve energy planning and avoid energy-related crises

The Growth Potential of <i>Bacillus cereus</i> in Ready-to-Reheat Vegetable Soups

Bacillus cereus (hereafter, B. cereus) poisoning often arises from the consumption of Ready-To-Reheat vegetable soups in which an intensive growth of the vegetative cells of B. cereus take place. The market for these soups is increasing significantly worldwide. For the producer it is important to determine if soups can promote the growth of B. cereus, by calculating its growth potential. We can achieve this goal by carrying out an efficient challenge test. In our study we have designed and performed a challenge test in three batches of an emmer (Triticum monococcum) and vegetable soup that undergo a second pasteurization treatment after packaging. We found out that under refrigeration conditions B. cereus is unable to multiply in the soup, instead, under conditions of thermal abuse, B. cereus can grow during 90 days of shelf life with a growth potential of 0.82 logarithms. It is essential to keep the entire production phase under control using effective GMP (Good Manufacturing Practices) and GHP (Good Hygiene Practices) measures, to ensure that the freshly produced soups contain low loads of the spores of B. cereus. In this way, the vegetative cells born from the germination of the spores cannot reach the infectious dose necessary to induce the food poisoning