Spatially Correlated Nuclear Magnetic Resonance Profiles as a Tool for Precision Agriculture.

Nuclear magnetic resonance (NMR) profiling, sample georeferentiaton, and geostatistics are applied to evaluate the spatial variability of metabolic expression of durum wheat in fields managed by precision agriculture. Durum wheat at three different vegetation stages, grown in two different places of the Basilicata region, in Italy, is analyzed by NMR. The spatial variability, within each field, of metabolites, quantified by NMR, is evidenced by appropriate geostatistic tools through the definition of a suitable metabolic index. Metabolic maps are compared to highlight the effects of soil and farming strategies.

Farming for Pharming: Novel Hydroponic Process in Contained Environment for Efficient Pharma-Grade Production of Saffron.

Soilless cultivation of saffron (Crocus sativus) in a controlled environment represents an interesting alternative to field cultivation, in order to obtain a standardized high-quality product and to optimize yields. In particular, pharma-grade saffron is fundamental for therapeutic applications of this spice, whose efficacy has been demonstrated in the treatment of macular diseases, such as Age-related Macular Degeneration (AMD). In this work, a hydroponic cultivation system was developed, specifically designed to meet the needs of C. sativus plant. Various cultivation recipes, different in spectrum and intensity of lighting, temperature, photoperiod and irrigation, have been adopted to study their effect on saffron production. The experimentation involved the cultivation of corms from two subsequent farm years, to identify and validate the optimal conditions, both in terms of quantitative yield and as accumulation of bioactive metabolites, with particular reference to crocins and picrocrocin, which define the 'pharma-grade' quality of saffron. Through HPLC analysis and chromatography it was possible to identify the cultivation parameters suitable for the production of saffron with neuroprotective properties, evaluated by comparison with an ISO standard and the REPRON® procedure. Furthermore, the biochemical characterization was completed through NMR and high-resolution mass spectrometry analyses of saffron extracts. The whole experimental framework allowed to establish an optimized protocol to produce pharma-grade saffron, allowing up to 3.2 g/m2 harvest (i.e., more than three times higher than field production in optimal conditions), which meets the standards of composition for the therapy of AMD.

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.

Optimised production of an anti-fungal antibody in Solanaceae hairy roots to develop new formulations against Candida albicans.

BACKGROUND: Infections caused by fungi are often refractory to conventional therapies and urgently require the development of novel options, such as immunotherapy. To produce therapeutic antibodies, a plant-based expression platform is an attractive biotechnological strategy compared to mammalian cell cultures. In addition to whole plants, hairy roots (HR) cultures can be used, representing an expression system easy to build up, with indefinite growth while handled under containment conditions. RESULTS: In this study the production in HR of a recombinant antibody, proved to be a good candidate for human immunotherapy against fungal infections, is reported. Expression and secretion of this antibody, in an engineered single chain (scFvFc) format, by HR from Nicotiana benthamiana and Solanum lycopersicum have been evaluated with the aim of directly using the deriving extract or culture medium against pathogenic fungi. Although both Solanaceae HR showed good expression levels (up to 68 mg/kg), an optimization of rhizosecretion was only obtained for N. benthamiana HR. A preliminary assessment to explain this result highlighted the fact that not only the presence of proteases, but also the chemical characteristics of the growth medium, can influence antibody yield, with implications on recombinant protein production in HR. Finally, the antifungal activity of scFvFc 2G8 antibody produced in N. benthamiana HR was evaluated in Candida albicans growth inhibition assays, evidencing encouraging results. CONCLUSIONS: Production of this anti-fungal antibody in HR of N. benthamiana and S. lycopersicum elucidated factors affecting pharming in this system and allowed to obtain promising ready-to-use immunotherapeutics against C. albicans.

Comparative analysis of plant-produced, recombinant dimeric IgA against cell wall ß-glucan of pathogenic fungi.

Immunoglobulins A (IgA) are crucially involved in protection of human mucosal surfaces from microbial pathogens. In this work, we devised and expressed in plants recombinant chimeric antifungal antibodies (Abs) of isotype A (IgA1, IgA2, and scFvFcA1), derived from a murine mAb directed to the fungal cell wall polysaccharide ß-glucan which had proven able to confer protection against multiple pathogenic fungi. All recombinant IgA (rIgA) were expressed and correctly assembled in dimeric form in plants and evaluated for yield, antigen-binding efficiency and antifungal properties in vitro, in comparison with a chimeric IgG1 version. Production yields and binding efficiency to purified ß-glucans showed significant variations not only between Abs of different isotypes but also between the different IgA formats. Moreover, only the dimeric IgA1 was able to strongly bind cells of the fungal pathogen Candida albicans and to restrain its adhesion to human epithelial cells. Our data indicate that IgG to IgA switch and differences in molecular structure among different rIgA formats can impact expression in plant and biological activity of anti-ß-glucans Abs and provide new insights for the design of recombinant IgA as anti-infective immunotherapeutics, whose potential is still poorly investigated.

High-density molecular characterization and association mapping in Ethiopian durum wheat landraces reveals high diversity and potential for wheat breeding.

Durum wheat (Triticum turgidum subsp. durum) is a key crop worldwide, and yet, its improvement and adaptation to emerging environmental threats is made difficult by the limited amount of allelic variation included in its elite pool. New allelic diversity may provide novel loci to international crop breeding through quantitative trait loci (QTL) mapping in unexplored material. Here, we report the extensive molecular and phenotypic characterization of hundreds of Ethiopian durum wheat landraces and several Ethiopian improved lines. We test 81 587 markers scoring 30 155 single nucleotide polymorphisms and use them to survey the diversity, structure, and genome-specific variation in the panel. We show the uniqueness of Ethiopian germplasm using a siding collection of Mediterranean durum wheat accessions. We phenotype the Ethiopian panel for ten agronomic traits in two highly diversified Ethiopian environments for two consecutive years and use this information to conduct a genome-wide association study. We identify several loci underpinning agronomic traits of interest, both confirming loci already reported and describing new promising genomic regions. These loci may be efficiently targeted with molecular markers already available to conduct marker-assisted selection in Ethiopian and international wheat. We show that Ethiopian durum wheat represents an important and mostly unexplored source of durum wheat diversity. The panel analysed in this study allows the accumulation of QTL mapping experiments, providing the initial step for a quantitative, methodical exploitation of untapped diversity in producing a better wheat.

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

BACKGROUND: 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. RESULTS: SSR markers specific to E. ventricosum were developed through pyrosequencing of an enriched genomic library. Primer pairs were designed for 217 microsatellites with a repeat size > 20 bp from 900 candidates. Primers were validated in parallel by in silico and in vitro PCR approaches. A total of 67 primer pairs successfully amplified specific loci and 59 showed polymorphism. A subset of 34 polymorphic SSR markers were used to study 70 both wild and cultivated enset accessions. A large number of alleles were detected along with a moderate to high level of genetic diversity. AMOVA revealed that intra-population allelic variations contributed more to genetic diversity than inter-population variations. UPGMA based phylogenetic analysis and Discriminant Analysis of Principal Components show that wild enset is clearly separated from cultivated enset and is more closely related to the out-group Musa spp. No cluster pattern associated with the geographical regions, where this crop is grown, was observed for enset landraces. Our results reaffirm the long tradition of extensive seed-sucker exchange between enset cultivating communities in Southern Ethiopia. CONCLUSION: The first set of genomic SSR markers were developed in enset. A large proportion of these markers were polymorphic and some were also transferable to related species of the genus Musa. This study demonstrated the usefulness of the markers in assessing genetic diversity and structure in enset germplasm, and provides potentially useful information for developing conservation and breeding strategies in enset.

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 complementary in situ and ex situ conservation strategies to foster conservation of enset genetic resources and associated indigenous knowledge system.

Plant production of anti-ß-glucan antibodies for immunotherapy of fungal infections in humans.

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 ß-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 ß-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially ß1,3-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.