A three-years survey of microbial contaminants in industrial hemp inflorescences from two Italian cultivation sites.

BACKGROUND: The use of industrial Cannabis sativa L. for recreational, cosmeceutical, nutraceutical, and medicinal purposes has gained momentum due to its rich content of valuable phytochemicals, such as cannabidiol (CBD) and cannabigerol (CBG). However, there are concerns regarding the risk of microbial contamination in plants grown outside controlled environments. Microbes associated with hemp can be either epiphytes or endophytes and may pose a risk of infectious illness for humans. METHODS: Seven Italian hemp genotypes, including Bernabeo, Carmagnola, Carmaleonte, Codimono, CS, Eletta Campana, and Fibranova, were cultivated in two distinct geographic locations, Catania and Rovigo, for three consecutive years from 2019 to 2021. Total aerobic microbes (TAMC), total combined yeasts/moulds (TYMC), the presence of bile-tolerant Gram-negative bacteria, and the absence of Escherichia coli and Salmonella spp. were evaluated and compared. The main phytocannabinoid content was measured and correlated with microbial contamination. RESULTS: Most samples analyzed in this study did not meet the European Pharmacopoeia microbiological limits. The detection of potential pathogens, such as E. coli and Salmonella spp., in the samples indicates that the use of inflorescences may represent a possible source of infection. Microbial contamination varied among harvesting seasons and production sites, with agroclimatic conditions influencing microbial load and composition. The presence of potentially pathogenic bacteria was less associated with seasonal climate variability and more likely affected by sporadic contamination from external sources. CBD concentration exhibited a negative correlation with bile-tolerant Gram-negative bacteria and total yeasts/moulds levels. Samples with lower CBD content were more contaminated than those with higher CBD levels, suggesting a potential protective effect of this phytochemical on the plant. CONCLUSIONS: The threshing residues (inflorescences, floral bracts, and leaves) of industrial hemp varieties represent a valuable product and a source of beneficial phytochemicals that warrants further exploration. While post-harvest sterilization methods may reduce microbiological risks, they may also degrade heat- and light-sensitive bioactive phytochemicals. The most promising strategy involves implementing best agronomic practices to maintain healthy and uncontaminated cultures. Rigorous monitoring and quality certification protocols are essential to mitigate the microbiological risk associated with the consumption of hemp-derived products.

Analysis of phytocannabinoids in hemp seeds, sprouts and microgreens.

Hemp-sprouts are emerging as a new class of attractive functional food due to their numerous health benefits when compared to other sprout species. Indeed, the high content of beneficial components including polyphenols and flavonoids makes this type of food a promising and successful market. However, the available literature on this topic is limited and often conflicting as regards to the content of phytocannabinoids. High-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) was applied in an untargeted metabolomics fashion to extracts of hemp seeds, sprouts and microgreens of nine different genotypes. Both unsupervised and supervised multivariate statistical analysis was performed to reveal variety-specific profiles of phytocannabinoids with surprisingly remarkable levels of phytocannabinoids even in chemotype V samples. Furthermore, a targeted HPLC-HRMS analysis was carried out for the quantitative determination of the major phytocannabinoids including CBDA, CBD, CBGA, CBG, CBCA, CBC, THCA, and trans-Δ9-THC. The last part of the study was focused on the evaluation of the enantiomeric composition of CBCA in hemp seeds, sprouts and microgreens in the different varieties by HPLC-CD (HPLC with online circular dichroism). Chiral analysis of CBCA showed a wide variability of its enantiomeric composition in the different varieties, thus contributing to the understanding of the intriguing stereochemical behavior of this compound in an early growth stage. However, further investigation is needed to determine the genetic factors responsible for the low enantiopurity of this compound.

Untargeted cannabinomics reveals the chemical differentiation of industrial hemp based on the cultivar and the geographical field location.

Cannabis sativa has long been harvested for industrial applications related to its fibers. Industrial hemp cultivars, a botanical class of Cannabis sativa with a low expression of intoxicating Δ9-tetrahydrocannabinol (Δ9-THC) have been selected for these purposes and scarcely investigated in terms of their content in bioactive compounds. Following the global relaxation in the market of industrial hemp-derived products, research in industrial hemp for pharmaceutical and nutraceutical purposes has surged. In this context, metabolomics-based approaches have proven to fulfill the aim of obtaining comprehensive information on the phytocompound profile of cannabis samples, going beyond the targeted evaluation of the major phytocannabinoids. In the present paper, an HRMS-based metabolomics study was addressed to seven distinct industrial hemp cultivars grown in four experimental fields in Northern, Southern, and Insular Italy. Since the role of minor phytocannabinoids as well as other phytocompounds was found to be critical in discriminating cannabis chemovars and in determining its biological activities, a comprehensive characterization of phytocannabinoids, flavonoids, and phenolic acids was carried out by LC-HRMS and a dedicated data processing workflow following the guidelines of the metabolomics Quality Assurance and Quality Control Consortium. A total of 54 phytocannabinoids, 134 flavonoids, and 77 phenolic acids were annotated, and their role in distinguishing hemp samples based on the geographical field location and cultivar was evaluated by ANOVA-simultaneous component analysis. Finally, a low-level fused model demonstrated the key role of untargeted cannabinomics extended to lesser-studied phytocompound classes for the discrimination of hemp samples.

Phytocannabinoids biosynthesis during early stages of development of young Cannabis sativa L. seedlings: Integrating biochemical and transcription data.

Cannabis sativa (L.) is characterized by great genetic and phenotypic diversity, also expressed in the array of bioactive compounds synthesized. Despite its great potential economic interest, knowledge of the biology and genetics of this crop is incomplete, and still many efforts are needed for a complete understanding of the molecular mechanisms regulating its key traits. To better understand the synthesis of these compounds, we analysed the transcription levels of cannabinoid pathway genes during early phases of plant development, then comparing the transcriptional results with a chemical characterization of the same samples. The work was conducted on both industrial and medicinal C. sativa plants, using samples belonging to three different chemotypes. Genes coding for the cannabinoid synthases, involved in the last step of the cannabinoid biosynthetic pathway, were found to be already expressed in the seed, providing a measure of the importance of this metabolism for the plant. Cannabichromenic acid is known as the first cannabinoid accumulating in the seedlings, shortly after emergence, and it was found that there is a good correspondence between transcript accumulation of the cannabichromenic acid synthase gene and accumulation of the corresponding metabolite.

When Cannabis sativa L. Turns Purple: Biosynthesis and Accumulation of Anthocyanins.

Environmental cues elicit anthocyanin synthesis in plant vegetative and reproductive tissues. Their accumulation in different organs accounts for their diverse biological functions, mainly related to their antioxidant properties, and it depends on a temporally and spatially regulated mechanism controlled by the action of a well-known multi-transcription factor complex. Despite the highly recognizable value of Cannabis sativa L. as a natural biorefinery of phytochemicals, very little information is known on anthocyanin pigmentation in this species. In this work, a targeted quantification of anthocyanins via HPLC-MS/MS, combined with the transcriptional profile via RT-qPCR of genes encoding for structural and decorating enzymes and regulatory transcription factors in different C. sativa tissues, help gain insights into the anthocyanin pathway in this species. To the best of our knowledge, this is the first report on the identification of cyanidin-3-rutinoside (keracyanin) as the major anthocyanin in C. sativa vegetative and floral tissues. Keracyanin amounts were higher than in small berries, suggesting that Cannabis biomass is a valuable source of colored antioxidants to be exploited in diverse applications. Furthermore, a gene putatively encoding for an anthocyanin DTX35 type transporter and CsTTG1 were identified in silico and their transcriptional levels were assessed via RT-qPCR. The results allow us to provide the first model of anthocyanin regulation in C. sativa, opening a new research scenario in this species for both breeding purposes and phytochemical exploitation.

Evaluation of the carotenoid and fat-soluble vitamin profile of industrial hemp inflorescence by liquid chromatography coupled to mass spectrometry and photodiode-array detection.

Industrial hemp (Cannabis sativa L.) is a plant matrix whose use is recently spreading for pharmaceutical and nutraceutical purposes. Detailed characterization of hemp composition is needed for future research that further exploits the beneficial effects of hemp compounds on human health. Among minor constituents, carotenoids and fat-soluble vitamins have largely been neglected to date despite carrying out several biological activities and regulatory functions. In the present paper, 22 target carotenoids and fat-soluble vitamins were analyzed in the inflorescences of seven Italian industrial hemp varieties cultivated outdoor. The analytes were extracted by cold saponification to avoid artifacts and analyzed by high-performance liquid chromatography coupled with Selected reaction monitoring mass spectrometry. Phytoene, phytofluene, and all-trans-ß-carotene were the most abundant in all analyzed samples (31-55 µg g-1, 11.6-29 µg g-1, and 7.3-53 µg g-1, respectively). Besides the target analytes, liquid chromatography coupled with photodiode-array detection allowed us to tentatively identify several other carotenoids based on their retention behavior and UV-vis spectra with the support of theoretical rules and data in the literature. To the best of our knowledge, this is the first comprehensive characterization of carotenoids and fat-soluble vitamins in industrial hemp inflorescence.

The B1080/B1192 molecular marker identifies hemp plants with functional THCA synthase and total THC content above legal limit.

In Cannabis sativa L. the presence of delta 9-tetrahydrocannabinolic acid (THCA) above legal limit is a challenging issue that still restricts the industrial exploitation of this promising crop. In recent years, the interest of entrepreneurs and growers who see hemp as a dynamic and profitable crop was joined by the growing knowledge on C. sativa genetics and genomics, accelerated by the application of high throughput tools. Despite the renewed interest in the species, much remains to be clarified, especially about the long-standing problem of THCA in hemp inflorescences, which could even result in the seizure of the whole harvest. Although several hypotheses have been formulated on the accumulation of this metabolite in industrial varieties, none is conclusive yet. In this work, individuals of a population of the hemp cultivar 'FINOLA' obtained from commercial seeds were investigated for total THC level and examined at molecular level. A marker linked to THCA synthase was found at a high incidence in both male and female plants, suggesting a considerable genetic variability within the seed batch. Full-length sequences encoding for putatively functional THCA synthases were isolated for the first time from the genome of both female and male plants of an industrial hemp variety and, using transcriptional analysis, the THCA synthase expression was quantified in mature inflorescences of individuals identified by the marker. Biochemical analyses finally demonstrated for these plants a 100% association between the predicted and actual chemotype.

Cis-Δ9-tetrahydrocannabinolic acid occurrence in Cannabis sativa L.

Cannabidiolic acid (CBDA) and trans-Δ9-tetrahydrocannabinolic acid (trans-Δ9-THCA) are known to be the major phytocannabinoids in Cannabis sativa L., along with their decarboxylated derivatives cannabidiol (CBD) and trans-Δ9-tetrahydrocannabinol (trans-Δ9-THC). The cis isomer of Δ9-THC has been recently identified, characterized and quantified in several Cannabis sativa varieties, which had been heated (decarboxylated) before the analysis. Since decarboxylation alters the original phytocannabinoids composition of the plant, this work reports the identification and characterization of the carboxylated precursor cis-Δ9-THCA. The compound was also synthesized and used as analytical standard for the development and validation of a liquid chromatography coupled to high resolution mass spectrometry-based method for its quantification in ten Cannabis sativa L. samples from different chemotypes. The highest concentrations of cis-Δ9-THCA were found in CBD-rich varieties, lower levels were observed in cannabigerol (CBG)-rich varieties (chemotype IV) and in those varieties with a balanced level of both CBD and THC (chemotype III), while its levels were not detectable in cannabichromene (CBC)-rich varieties (chemotype VI). The presence of the cis isomer of THC and THCA raises the question on whether to include or not this species in the calculation of the total amount of THC to classify a cannabis variety as a drug-type or a fiber-type (hemp).

Does Plant Breeding for Antioxidant-Rich Foods Have an Impact on Human Health?

Given the general beneficial effects of antioxidants-rich foods on human health and disease prevention, there is a continuous interest in plant secondary metabolites conferring attractive colors to fruits and grains and responsible, together with others, for nutraceutical properties. Cereals and Solanaceae are important components of the human diet, thus, they are the main targets for functional food development by exploitation of genetic resources and metabolic engineering. In this review, we focus on the impact of antioxidants-rich cereal and Solanaceae derived foods on human health by analyzing natural biodiversity and biotechnological strategies aiming at increasing the antioxidant level of grains and fruits, the impact of agronomic practices and food processing on antioxidant properties combined with a focus on the current state of pre-clinical and clinical studies. Despite the strong evidence in in vitro and animal studies supporting the beneficial effects of antioxidants-rich diets in preventing diseases, clinical studies are still not sufficient to prove the impact of antioxidant rich cereal and Solanaceae derived foods on human.

Kynurenine and kynurenic acid: Two human neuromodulators found in Cannabis sativa L.

L-Kynurenine (KYN) and kynurenic acid (KYNA) are products of the metabolism of L-tryptophan (TRP) in the central nervous system of animals, but they are not commonly found in plants. In particular, KYNA is known for its interesting pharmacological properties (anti-oxidative, anti-inflammatory, hypolipidemic, and neuroprotective), which suggest a potential functional food ingredient role. The three compounds were identified in samples of Cannabis sativa L. by means of high-performance liquid chromatography coupled to high-resolution mass spectrometry using an untargeted metabolomics approach. Their concentrations were evaluated using a targeted metabolomics method in three organs of the plant (roots, stem, and leaves) in soil at two different growth stages and in hydroponics conditions. The distribution of TRP, KYN and KYNA was found tendentially higher in leaves compared to stem and roots and changed over time. Moreover, the levels of KYNA found in this study are unprecedentedly high compared to those found so far in other plant species, suggesting that Cannabis sativa L. could be a promising alternative source of this metabolite.

Genome-Wide Identification, Characterization and Expression Pattern Analysis of the γ-Gliadin Gene Family in the Durum Wheat (Triticum durum Desf.) Cultivar Svevo.

Very recently, the genome of the modern durum wheat cv. Svevo was fully sequenced, and its assembly is publicly available. So, we exploited the opportunity to carry out an in-depth study for the systematic characterization of the γ-gliadin gene family in the cv. Svevo by combining a bioinformatic approach with transcript and protein analysis. We found that the γ-gliadin family consists of nine genes that include seven functional genes and two pseudogenes. Three genes, Gli-γ1a, Gli-γ3a and Gli-γ4a, and the pseudogene Gli-γ2a* mapped on the A genome, whereas the remaining four genes, Gli-γ1b, Gli-γ2b, Gli-γ3b and Gli-γ5b, and the pseudogene Gli-γ4b* mapped on the B genome. The functional γ-gliadins presented all six domains and eight-cysteine residues typical of γ-gliadins. The Gli-γ1b also presented an additional cysteine that could possibly have a role in the formation of the gluten network through binding to HMW glutenins. The γ-gliadins from the A and B genome differed in their celiac disease (CD) epitope content and composition, with the γ-gliadins from the B genome showing the highest frequency of CD epitopes. In all the cases, almost all the CD epitopes clustered in the central region of the γ-gliadin proteins. Transcript analysis during seed development revealed that all the functional γ-gliadin genes were expressed with a similar pattern, although significant differences in the transcript levels were observed among individual genes that were sometimes more than 60-fold. A progressive accumulation of the γ-gliadin fraction was observed in the ripening seeds that reached 34% of the total gliadin fraction at harvest maturity. We believe that the insights generated in the present study could aid further studies on gliadin protein functions and future breeding programs aimed at the selection of new healthier durum wheat genotypes.

The novel heptyl phorolic acid cannabinoids content in different Cannabis sativa L. accessions.

The recent discovery of the novel heptyl phytocannabinoids cannabidiphorol (CBDP) and Δ9-tetrahydrocannabiphorol (Δ9-THCP) raised a series of questions relating to the presence and abundance of these new unorthodox compounds in cannabis inflorescence or derived products. As fresh inflorescence contains mainly their acid precursors, which are not commercially available, an ad hoc stereoselective synthesis was performed in order to obtain cannabidiphorolic acid (CBDPA) and Δ9-tetrahydrocannabiphorolic acid (THCPA) to be used as analytical standards for quantitative purposes. The present work reports an unprecedented targeted analysis of both pentyl (C5) and heptyl (C7) CBD- and THC-type compounds in forty-nine cannabis samples representing four different chemotypes. Moreover, the ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry-based method was applied for the putative identification of other heptyl homologs of the most common phytocannabinoid acids, including cannabigerophorolic acid (CBGPA), cannabichromephorolic acid (CBCPA), cannabinophorolic acid (CBNPA), cannabielsophorolic acid (CBEPA), cannabicyclophorolic acid (CBLPA), cannabitriophorolic acid (CBTPA), and cannabiripsophorolic acid (CBRPA).

Analysis of Sequence Variability and Transcriptional Profile of Cannabinoid synthase Genes in Cannabis sativa L. Chemotypes with a Focus on Cannabichromenic acid synthase.

Cannabis sativa L. has been long cultivated for its narcotic potential due to the accumulation of tetrahydrocannabinolic acid (THCA) in female inflorescences, but nowadays its production for fiber, seeds, edible oil and bioactive compounds has spread throughout the world. However, some hemp varieties still accumulate traces of residual THCA close to the 0.20% limit set by European Union, despite the functional gene encoding for THCA synthase (THCAS) is lacking. Even if some hypotheses have been produced, studies are often in disagreement especially on the role of the cannabichromenic acid synthase (CBCAS). In this work a set of European Cannabis genotypes, representative of all chemotypes, were investigated from a chemical and molecular point of view. Highly specific primer pairs were developed to allow an accurate distinction of different cannabinoid synthases genes. In addition to their use as markers to detect the presence of CBCAS at genomic level, they allowed the analysis of transcriptional profiles in hemp or marijuana plants. While the high level of transcription of THCAS and cannabidiolic acid synthase (CBDAS) clearly reflects the chemical phenotype of the plants, the low but stable transcriptional level of CBCAS in all genotypes suggests that these genes are active and might contribute to the final amount of cannabinoids.

Essential Oil of Cannabis sativa L: Comparison of Yield and Chemical Composition of 11 Hemp Genotypes.

Cannabis sativa L. is an annual species cultivated since antiquity for different purposes. While, in the past, hemp inflorescences were considered crop residues, at present, they are regarded as valuable raw materials with different applications, among which extraction of the essential oil (EO) has gained increasing interest in many fields. The aim of the present study is the evaluation of the yield and the chemical composition of the EO obtained by hydrodistillation from eleven hemp genotypes, cultivated in the same location for two consecutive growing seasons. The composition of the EOs was analyzed by GC-MS, and then subjected to multivariate statistical analysis. Sesquiterpenes represented the main class of compounds in all the EOs, both in their hydrocarbon and oxygenated forms, with relative abundances ranging from 47.1 to 78.5%; the only exception was the Felina 32 sample collected in 2019, in which cannabinoids predominated. Cannabinoids were the second most abundant class of compounds, of which cannabidiol was the main one, with relative abundances between 11.8 and 51.5%. The statistical distribution of the samples, performed on the complete chemical composition of the EOs, evidenced a partition based on the year of cultivation, rather than on the genotype, with the exception of Uso-31. Regarding the extraction yield, a significant variation was evidenced among both the genotypes and the years of cultivation.

Phytocannabinomics: Untargeted metabolomics as a tool for cannabis chemovar differentiation.

Cannabis sativa is traditionally classified according to five chemotypes based on the concentration of the main phytocannabinoids tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabigerol (CBG). However, cannabis chemovars and varieties very often present similar concentrations of such phytocannabinoids but different chemical profiles, which is unavoidably translated into different pharmacological effects when used for therapeutic purposes. For this reason, a more refined approach is needed for chemovar distinction, which is described in this study and named phytocannabinomics. The classification was achieved by a comprehensive characterization of the phytocannabinoid composition, by liquid chromatography coupled to high-resolution mass spectrometry untargeted metabolomics for the detection of over a hundred phytocannabinoids, and data analysis by chemometrics for chemovars differentiation. The method was developed on fifty cannabis varieties, grown under the same conditions, and was validated to discriminate between the standard chemotypes by partial least squares discriminant analysis. Then, the method was extended to consider the entire chemical variety of the cannabis accessions, by an unsupervised approach based on the principal component analysis. The latter approach clearly indicated several new subgroups within the traditional classifications, which arise from a unique composition of the minor phytocannabinoids. The existence of these subgroups, which were never described before, is of critical importance for evaluating the pharmacological effects of cannabis chemovars.

In Silybum marianum Italian wild populations the variability of silymarin profiles results from the combination of only two stable chemotypes.

Silybum marianum (L.) Gaertn. is an important medicinal plant belonging to Mediterranean flora. The medicinal properties of the species are mainly due to silymarin, a combination of different flavonolignans contained in the fruit. As for silymarin, so far a wide variability of possible S. marianum chemotypes has been described. In the present study the flavonolignan profile of 40 different S. marianum wild accessions was analysed at both population and single plant level, further extending the analysis to progenies derived from crosses between parental lines with different chemotypes. The results of this work indicate that S. marianum wild populations can be composed either of individuals with the same chemotype, or heterogeneous mixtures of individuals characterized by different chemotypes. Only three chemotypes (A, B and C) have been identified among Italian wild populations. Based on data collected we furthermore propose that chemotype C is the result of the hybridization between A and B chemotypes. If assessed at single plant level, chemotypes are extremely stable therefore evidencing a strong genetic control of silymarin biosynthetic pathway. Chemotypes A and B are present in all the analysed regions and no clear correlation between chemotypes and geographic features has been found. In conclusion, this work provides a general procedure for the characterization of different and stable chemotypes, for a deeper understanding of silymarin biosynthetic pathway, and in order to implement S. marianum breeding programmes aiming to improve silymarin quality.

Selection and validation of reference genes for RT-qPCR normalization in different tissues of milk thistle (Silybum marianum, Gaert.).

Quantitative reverse transcription PCR is a sensitive technique for evaluating transcriptional profiles in different experimental datasets. To obtain a reliable quantification of the transcripts level, data normalization with stable reference genes is required. Stable reference genes are identified after analysis of their transcripts profile in every new experiment and species of interest. In Silybum marianum, a widely cultivated officinal plant, only few gene expression studies exist, and reference genes for RT-qPCR studies in the diverse plant tissues have never been investigated before. In this work, the expression stability of 10 candidate reference genes was evaluated in leaves, roots, stems and fruits of S. marianum grown under physiological environmental condition. The stability values for each candidate reference gene were calculated by four canonical statistical algorithms GeNorm, NormFinder, Bestkeeper and ΔCt method in different subsets of samples, then they were ranked with RefFinder from the most to the least suitable for normalization. Best combinations of reference genes are finally proposed for different experimental data sets, including all tissues, vegetative, and reproductive tissues separately. Three target genes putatively involved in important biosynthetic pathway leading to key metabolites in the fruits of milk thistle, such as silymarin and fatty acids, were analyzed with the chosen panels of reference genes, in comparison to the ones used in previous papers. To the best of our knowledge, this is the first report on a reliable and systematic identification and validation of the reference genes for RT-qPCR normalization to study gene expression in S. marianum.

In Silico Identification of MYB and bHLH Families Reveals Candidate Transcription Factors for Secondary Metabolic Pathways in Cannabis sativa L.

Plant secondary metabolic pathways are finely regulated by the activity of transcription factors, among which members of the bHLH and MYB subfamilies play a main role. Cannabis sativa L. is a unique officinal plant species with over 600 synthesized phytochemicals having diverse scale-up industrial and pharmaceutical usage. Despite comprehensive knowledge of cannabinoids' metabolic pathways, very little is known about their regulation, while the literature on flavonoids' metabolic pathways is still scarce. In this study, we provide the first genome-wide analysis of bHLH and MYB families in C. sativa reference cultivar CBDRx and identification of candidate coding sequences for these transcription factors. Cannabis sativa bHLHs and MYBs were then classified into functional subfamilies through comparative phylogenetic analysis with A. thaliana transcription factors. Analyses of gene structure and motif distribution confirmed that CsbHLHs and CsMYBs belonging to the same evolutionary clade share common features at both gene and amino acidic level. Candidate regulatory genes for key metabolic pathways leading to flavonoid and cannabinoid synthesis in Cannabis were also retrieved. Furthermore, a candidate gene approach was used to identify structural enzyme-coding genes for flavonoid and cannabinoid synthesis. Taken as a whole, this work represents a valuable resource of candidate genes for further investigation of the C. sativa cannabinoid and flavonoid metabolic pathways for genomic studies and breeding programs.

Selected isolates of Trichoderma gamsii induce different pathways of systemic resistance in maize upon Fusarium verticillioides challenge.

The pink ear rot is one of the most damaging maize diseases, caused by the mycotoxigenic fungal pathogen, Fusarium verticillioides. The application of biological control agents, like antagonistic and/or resistance inducer microorganisms, is an option to reduce fungal infection and kernel contamination in a sustainable and environmentally friendly way. It is well known that Trichoderma species are non-pathogenic fungi able to antagonize plant pathogens and to induce systemic resistance in plants. The present work aimed to verify if Trichoderma spp., applied to maize kernels, affect the plant growth and induce systemic responses to F. verticillioides. Besides, the capability to reduce fumonisin concentration in liquid cultures was investigated. Two T. gamsii (IMO5 and B21), and one T. afroharzianum (B75) isolates, selected both for antagonism and for the ability to reduce root infections, significantly reduced the endophytic development of the stem-inoculated pathogen, compared to the control. The mechanisms of action appeared to be strain-specific, with IMO5 enhancing transcript levels of marker genes of Induced Systemic Resistance (ZmLOX10, ZmAOS, and ZmHPL) while B21 enhancing marker genes of Systemic Acquired Resistance (ZmPR1 and ZmPR5), as evinced by measuring their expression profiles in the leaves. Moreover, IMO5 promoted plant growth, while B21 was able to significantly reduce the fumonisin content in a liquid medium. The results of this work give new evidence that the seed application of T. gamsii is a promising tool for controlling F. verticillioides to be integrated with breeding and the adoption of good agricultural practices.

Comparative analysis of allergen genes and pro-inflammatory factors in pollen and fruit of apple varieties.

Allergy to freshly consumed apple fruits is often associated to pollinosis and manifested as oral allergy syndrome (OAS). The allergenic properties of apple varieties differ greatly, spanning from low allergenic to high allergenic varieties. The knowledge of the genetic determinants for allergenicity has been of great interest in scientific community for several years, but the molecular mechanisms involved are still little understood. Here, factors putatively involved in allergenicity were investigated at biochemical and molecular level in pollen and in fruits of apple varieties differing in their allergenic potential. Among putative sensitizing factors, transglutaminase (TGase) and phospholipase A2 (PLA2) were considered together with reactive oxygen species (ROS) and known apple allergen genes, with particular attention devoted to the Mal d 1 gene family, the most important one in sensitization. We found that the expression of some allergen genes and the activities of TGase, PLA2 and ROS producing enzyme are lower in the hypo-allergenic variety 'Durello di Forlì' in comparison with the high-allergenic genotypes 'Gala' and 'Florina'. These results highlight correlations among allergen expressions, enzymatic activities and apple cultivars; these data underline the possibility that some of them could be used in the future as markers for allergenicity.