Several secondary metabolite gene clusters in the genomes of ten Penicillium spp. raise the risk of multiple mycotoxin occurrence in chestnuts.

Penicillium spp. produce a great variety of secondary metabolites, including several mycotoxins, on food substrates. Chestnuts represent a favorable substrate for Penicillium spp. development. In this study, the genomes of ten Penicillium species, virulent on chestnuts, were sequenced and annotated: P. bialowiezense. P. pancosmium, P. manginii, P. discolor, P. crustosum, P. palitans, P. viridicatum, P. glandicola, P. taurinense and P. terrarumae. Assembly size ranges from 27.5 to 36.8 Mb and the number of encoded genes ranges from 9,867 to 12,520. The total number of predicted biosynthetic gene clusters (BGCs) in the ten species is 551. The most represented families of BGCs are non ribosomal peptide synthase (191) and polyketide synthase (175), followed by terpene synthases (87). Genome-wide collections of gene phylogenies (phylomes) were reconstructed for each of the newly sequenced Penicillium species allowing for the prediction of orthologous relationships among our species, as well as other 20 annotated Penicillium species available in the public domain. We investigated in silico the presence of BGCs for 10 secondary metabolites, including 5 mycotoxins, whose production was validated in vivo through chemical analyses. Among the clusters present in this set of species we found andrastin A and its related cluster atlantinone A, mycophenolic acid, patulin, penitrem A and the cluster responsible for the synthesis of roquefortine C/glandicoline A/glandicoline B/meleagrin. We confirmed the presence of these clusters in several of the Penicillium species conforming our dataset and verified their capacity to synthesize them in a chestnut-based medium with chemical analysis. Interestingly, we identified mycotoxin clusters in some species for the first time, such as the andrastin A cluster in P. flavigenum and P. taurinense, and the roquefortine C cluster in P. nalgiovense and P. taurinense. Chestnuts proved to be an optimal substrate for species of Penicillium with different mycotoxigenic potential, opening the door to risks related to the occurrence of multiple mycotoxins in the same food matrix.

Powdery mildew caused by Erysiphe corylacearum: An emerging problem on hazelnut in Italy.

Erysiphe corylacearum has recently been reported in northern Italy (Piedmont) and other European countries as the causal agent of a new emerging powdery mildew on hazelnut. This disease is much more dangerous than the common hazelnut powdery mildew caused by Phyllactinia guttata as it significantly reduces yield and quality of hazelnuts. This study aimed to perform morphological and molecular characterization of the fungal isolates from powdery mildew-infected plants in the Piedmont Italian region. Additionally, genetic diversity studies and pathogenicity tests were conducted. Thirty-six fungal isolates originating from symptomatic hazelnut plants exhibiting specific powdery mildew symptoms on the superior leaf side were identified morphologically as E. corylacearum. Single- and multilocus sequence typing of five loci (ITS, rpb2, CaM, GAPDH and GS) assigned all isolates as E. corylacearum. Multilocus and GAPDH phylogenetic studies resulted in the most efficient characterization of E. corylacearum. Studied fungal isolates were able to cause new emerging powdery mildew disease by fulfilling Koch's postulates. The emergence of powdery mildew disease in Italy revealed the E. corylacearum subgrouping, population expansion, and high nucleotide similarity with other recently identified E. corylacearum hazelnut isolates. To contain this harmful disease and inhibit the fungus spread into new geographical zones, it will be necessary to implement more rigorous monitoring in neighboring hazelnut plantations near infected hazelnuts, use sustainable fungicides and search for new biocontrol agents.

A haplotype-resolved chromosome-level assembly and annotation of European hazelnut (C. avellana cv. Jefferson) provides insight into mechanisms of eastern filbert blight resistance.

European hazelnut (Corylus avellana L.) is an important tree nut crop. Hazelnut production in North America is currently limited in scalability due to Anisogramma anomala, a fungal pathogen that causes Eastern Filbert Blight (EFB) disease in hazelnut. Successful deployment of EFB resistant cultivars has been limited to the state of Oregon, where the breeding program at Oregon State University (OSU) has released cultivars with a dominant allele at a single resistance locus identified by classical breeding, linkage mapping, and molecular markers. C. avellana cultivar "Jefferson" is resistant to the predominant EFB biotype in Oregon and has been selected by the OSU breeding program as a model for hazelnut genetic and genomic research. Here, we present a near complete, haplotype-resolved chromosome-level hazelnut genome assembly for "Jefferson". This new assembly is a significant improvement over a previously published genome draft. Analysis of genomic regions linked to EFB resistance and self-incompatibility confirmed haplotype splitting and identified new gene candidates that are essential for downstream molecular marker development, thereby facilitating breeding efforts.

Mycotoxin Contamination in Hazelnut: Current Status, Analytical Strategies, and Future Prospects.

Hazelnuts represent a potential source of mycotoxins that pose a public health issue due to their increasing consumption as food ingredients worldwide. Hazelnuts contamination by mycotoxins may derive from fungal infections occurring during fruit development, or in postharvest. The present review considers the available data on mycotoxins detected in hazelnuts, on fungal species reported as infecting hazelnut fruit, and general analytical approaches adopted for mycotoxin investigation. Prompted by the European safety regulation concerning hazelnuts, many analytical methods have focused on the determination of levels of aflatoxin B1 (AFB1) and total aflatoxins. An overview of the available data shows that a multiplicity of fungal species and further mycotoxins have been detected in hazelnuts, including anthraquinones, cyclodepsipeptides, ochratoxins, sterigmatocystins, trichothecenes, and more. Hence, the importance is highlighted in developing suitable methods for the concurrent detection of a broad spectrum of these mycotoxins. Moreover, control strategies to be employed before and after harvest in the aim of controlling the fungal contamination, and in reducing or inactivating mycotoxins in hazelnuts, are discussed.

Systemic effects of Tuber melanosporum inoculation in two Corylus avellana genotypes.

Roots of the European hazelnut (Corylus avellana L.), i.e., one of the most economically important nut species, form symbiosis with ectomycorrhizal (ECM) fungi, including truffles. Although physical interactions only occur in roots, the presence of mycorrhizal fungi can lead to metabolic changes at a systemic level, i.e., in leaves. However, how root colonization by ECM fungi modifies these processes in the host plant has so far not been widely studied. This work aimed to investigate the response in two C. avellana genotypes, focusing on leaves from plants inoculated with the black truffle Tuber melanosporum Vittad. Transcriptomic profiles of leaves of colonized plants were compared with those of non-colonized plants, as well as sugar and polyphenolic content. Results suggested that T. melanosporum has the potential to support plants in stressed conditions, leading to the systemic regulation of several genes involved in signaling and defense responses. Although further confirmation is needed, our results open new perspectives for future research aimed to highlight novel aspects in ECM symbiosis.

Ecology of Diaporthe eres, the causal agent of hazelnut defects.

Diaporthe eres has been recently reported as the causal agent of hazelnut defects, with characteristic brown spots on the kernels surface and internal fruit discoloration. Knowledge regarding the ecology of this fungus is poor but, is critical to support a rationale and effective hazelnut crop protection strategy. Therefore, a study was performed to describe and model the effect of different abiotic factors such as temperature (T, 5-35°C, step 5°C) and water activity (aw 0.83-0.99, step 0.03) regimes on D. eres mycelial growth, pycnidial conidiomata development and asexual spore production during a 60-day incubation period. Alpha conidia germination was tested in the same T range and at different relative humidities (RH = 94, 97 and 100%) over 48 h incubation period. Fungal growth was observed from the first visual observation; regarding pycnidia and cirrhi, their development started after 8 and 19 days of incubation, respectively and increased over time. The optimum T for growth was 20-25°C and for pycnidia and cirrhi development was 30°C; aw ≥ 0.98 was optimal for the tested steps of the fungal cycle. The best condition for conidial germination of D. eres was at 25°C with RH = 100%. Quantitative data obtained were fitted using non- linear regression functions (Bete, logistic and polynomial), which provided a very good fit of the biological process (R2 = 0.793-0.987). These functions could be the basis for the development of a predictive model for the infection of D. eres of hazelnuts.

Phylogeny and taxonomy of powdery mildew caused by Erysiphe species on Corylus hosts.

Erysiphe species (powdery mildews) on Corylus and Ostrya hosts (Betulaceae subfam. Coryloideae) in Asia and North America are widespread pathogens on these economically and ecologically valuable nut crops. An improved understanding of their phylogeny and taxonomy is of ecological and applied importance. Phylogenetic analyses and morphological reexaminations conducted in this study revealed a higher degree of diversity and cryptic speciation than reflected in earlier species concepts. North American collections on C. cornuta, which were previously assigned to E. corylacearum, proved to constitute a species of its own and are herein introduced as E. cornutae, sp. nov. Two additional North American species, E. coryli-americanae, sp. nov. and E. ostryae, sp. nov., have been detected on C. americana and O. virginiana and are described. They are morphologically similar to E. cornutae, but genetically distinct. Based on phylogenetic analyses, E. corylacearum is an Asian species confined to various Asian Corylus species. Sequence data retrieved from Japanese type material of E. corylicola revealed that this species clusters with sequences from E. elevata on Catalpa species, distant from all other Erysiphe species on Corylus. Morphologically similar, yet distinct, specimens on C. sieboldiana, which were previously assigned to E. corylicola, form a distinct, distant clade. The species involved is described herein as E. pseudocorylacearum, sp. nov. Additionally, an unusual infection of C. sieboldiana in Japan by E. syringae has been shown by means of sequence data. The phylogeny and taxonomy of Erysiphe species belonging to the Corylioideae are discussed in detail, and a key to the species concerned is provided.

Survey of Salmonella in raw tree nuts at retail in the United States.

The objective of this survey was to estimate the prevalence, contamination level, and genetic diversity of Salmonella in selected raw, shelled tree nuts (Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios, and walnuts) at retail markets in the United States. A total of 3,374 samples of eight tree nuts were collected from different types of retail stores and markets nationwide between September 2015 and March 2017. These samples (375 g) were analyzed using a modified FDA's BAM Salmonella culture method. Of the 3,374 samples, 15 (0.44%) (95% confidence interval [CI] [0.25, 0.73]) were culturally confirmed as containing Salmonella; 17 isolates were obtained. Among these isolates, there were 11 serotypes. Salmonella was not detected in Brazil nuts (296), hazelnuts (487), pecans (510), pine nuts (500), and walnuts (498). Salmonella prevalence estimates in cashews (510), macadamia (278), and pistachios (295) were 0.20% (95% CI [<0.01, 1.09]), 2.52% (95% CI [1.02, 5.12]), and 2.37% (95% CI [0.96, 4.83]), respectively. The rates of Salmonella isolation from major/big-chain supermarkets (1381), small-chain supermarkets (328), discount/variety/drug stores (1329), and online (336) were 0.29% (95% CI [0.08, 0.74]), 0.30% (95% CI [0.01, 1.69]), 0.45% (95% CI [0.17, 0.98]), and 1.19% (95% CI [0.33, 3.02]), respectively. Salmonella prevalence in organic (530) and conventional (2,844) nuts was not different statistically (P = 0.0601). Of the enumerated samples (15), 80% had Salmonella levels ≤0.0092 most probable number (MPN)/g. The highest contamination level observed was 0.75 MPN/g. The prevalence and contamination levels of Salmonella in the tree nuts analyzed were generally comparable to previous reports. Pulsed-field gel electrophoresis, serotype, and sequencing data all demonstrated that Salmonella population in nuts is very diverse genetically. PRACTICAL APPLICATION: The prevalence, contamination level, and genetic diversity of Salmonella in eight types of tree nuts (3,374 samples collected nationwide) revealed in this survey could help the development of mitigation strategies to reduce public health risks associated with consumption of these nuts.

Identification of native endophytic Trichoderma spp. for investigation of in vitro antagonism towards Armillaria mellea using synthetic- and plant-based substrates.

AIMS: To isolate endophytic Trichoderma species and investigate the potential for biological control of the root rot pathogen Armillaria mellea. METHODS AND RESULTS: In all, 40 Trichoderma isolates were obtained from a range of host plants and identities were confirmed by ITS, rpb2 and tef1 sequence. When tested in dual culture assays for antagonism against A. mellea, Trichoderma isolates overgrew the A. mellea colonies within four days and by eight days 38 Trichoderma isolates significantly reduced A. mellea colony size. Armillaria mellea was unable to be recovered from five of eight co-cultivations tested, suggesting Trichoderma had killed the A. mellea in these cases. Pre-colonized hazel disks were used to determine what happens in a more heterogeneous situation with A. mellea and a refined set of eight Trichoderma isolates. Similar to plate-based assays, Trichoderma quickly covered A. mellea stopping any further growth and two Trichoderma isolates were able to eradicate A. mellea. CONCLUSIONS: Of the Trichoderma spp. tested, endophytic isolates of Trichoderma virens and T. hamatum offered the greatest antagonism towards A. mellea. Using pre-colonized hazel disks was of great importance for this work to demonstrate the fungal interactions in plant material. SIGNIFICANCE AND IMPACT OF THE STUDY: Controlling Armillaria root rot is difficult with chemical treatments, thus an environmentally benign and cost-effective alternative is required. This study highlights the prospect of biological control as an effective, environmentally friendly alternative to chemicals.

Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillusflavus and A. parasiticus in Hazelnut.

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100-999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.

Streptomyces coryli sp. nov., isolated from hazelnut orchard soil.

A novel actinobacteria, isolate A7024T, was isolated from commercial hazelnut orchard soil sample which was collected at Duzce, West Black Sea region, Turkey. A polyphasic taxonomic study was carried out to determine the status of this isolate. The phylogenetic tree reconstructed using the neighbour-joining algorithm based on 16S rRNA gene sequences indicated that isolate A7024T was positioned within the members of the genus Streptomyces with the highest sequence similarity (97.7 %) to Streptomyces cadmiisoli ZFG47T. The organism formed an extensively branched substrate and aerial hyphae which generated irregular rod-shaped spores with smooth-surfaces. The cell wall of strain A7024T contained ll-diaminopimelic. Glucose, mannose and ribose were detected as whole-cell sugars. Its polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, three unidentified phospholipids and three unidentified glycolipids. Major menaquinones were MK-9(H6) and MK-9(H4). The major cellular fatty acids were iso-C16 : 0, anteiso-C15 : 0 and anteiso-C17 : 0. Strain A7024T had a genome size of 9.0 Mb with a genome G+C content of 71.5 mol%. The low level of 16S rRNA gene similarity, 19.3 ± 2.3% digital DNA-DNA hybridization and 76.94 % average nucleotide identity values, as well as some different phenotypic characteristics allowed the strain to be distinguished from the closely related type strains. Therefore, it is concluded that strain A7024T represents a novel species of the genus of Streptomyces, for which the name Streptomyces coryli sp. nov. is proposed. The type strain is A7024T (=DSM 42066T=KCTC 29102T=NRRL B-24888T).

Whole fungal elicitors boost paclitaxel biosynthesis induction in Corylus avellana cell culture.

Paclitaxel is an effective natural-source chemotherapeutic agent commonly applied to treat a vast range of cancers. In vitro Corylus avellana culture has been reported as a promising and inexpensive system for paclitaxel production. Fungal elicitors have been made known as the most efficient strategy for the biosynthesis induction of secondary metabolites in plant in vitro culture. In this research, C. avellana cell suspension culture (CSC) was exposed to cell extract (CE) and culture filtrate (CF) derived from Camarosporomyces flavigenus, either individually or combined treatment, in mid and late log phase. There is no report on the use of whole fungal elicitors (the combined treatment of CE and CF) for the elicitation of secondary metabolite biosynthesis in plant in vitro culture. The combined treatment of CE and CF significantly led to more paclitaxel biosynthesis and secretion than the individual use of them. Also, multivariate statistical approaches including stepwise regression (SR), ordinary least squares regression (OLSR), principal component regression (PCR) and partial least squares regression (PLSR) were used to model and predict paclitaxel biosynthesis and secretion. Based on value account for (VAF), root mean square error (RMSE), coefficient of determination (R2), mean absolute percentage error (MAPE) and relative percent difference (RPD) can be concluded that mentioned regression models effectively worked only for modeling and predicting extracellular paclitaxel portion in C. avellana cell culture.

HPLC-MS/MS Method for the Detection of Selected Toxic Metabolites Produced by Penicillium spp. in Nuts.

Penicillium spp. are emerging as producers of mycotoxins and other toxic metabolites in nuts. A HPLC-MS/MS method was developed to detect 19 metabolites produced by Penicillium spp. on chestnuts, hazelnuts, walnuts and almonds. Two extraction methods were developed, one for chestnuts and one for the other three nuts. The recovery, LOD, LOQ and matrix effect were determined for each analyte and matrix. Correlation coefficients were always >99.99%. In walnuts, a strong signal suppression was observed for most analytes and patulin could not be detected. Six strains: Penicillium bialowiezense, P. brevicompactum, P. crustosum, P. expansum, P. glabrum and P. solitum, isolated from chestnuts, were inoculated on four nuts. Chestnuts favored the production of the largest number of Penicillium toxic metabolites. The method was used for the analysis of 41 commercial samples: 71% showed to be contaminated by Penicillium-toxins. Cyclopenin and cyclopenol were the most frequently detected metabolites, with an incidence of 32% and 68%, respectively. Due to the risk of contamination of nuts with Penicillium-toxins, future studies and legislation should consider a larger number of mycotoxins.

Identity and pathogenicity of some fungi associated with hazelnut (Corylus avellana L.) trunk cankers in Oregon.

Four fungi isolated from trunks and branches of European hazelnut (Corylus avellana L.) from commercial orchards in the Willamette Valley, Oregon were characterized and pathogenicity was tested on potted hazelnut trees. The acreage of hazelnuts in Oregon has expanded greatly in recent years in response to the availability of Eastern filbert blight resistant cultivars. Fungi were characterized using the BLASTn algorithm and the GenBank database with multiple partial gene sequence(s). If BLASTn and GenBank were not sufficient for species-level identification, then a multilocus sequence analysis (MLSA) was performed. The four pathogens were identified as Diplodia mutilla (Fr.) Mont., Dothiorella omnivora B.T. Linaldeddu, A. Deidda & B. Scanu, Valsa cf. eucalypti Cooke & Harkn., and Diaporthe eres Nitschke. All pathogens but D. omnivora have not been previously reported from European hazelnut in the literature. All four pathogens caused lesions on trunks bare root hazelnut trees cv. 'Jefferson' planted in pots in the greenhouse and fungi were re-isolated from inoculated trees. D. mutilla appeared particularly aggressive in repeated inoculation experiments.

Signal pattern plot: a simple tool for time-dependent metabolomics studies by 1H NMR spectroscopy.

We show an alternative way to visualize time course NMR data without the application of multivariate data analysis, based on the temporal change of the metabolome of hazelnuts after mold infestation. Fresh hazelnuts were inoculated with eight different natural mold species and the growth was studied over a period of 14 days. The data were plotted in a color-coded scheme showing metabolic changes as a function of chemical shift, which we named signal pattern plot. This plot graphically displays alteration (trend) of a respected signal over time and allows visual interpretation in a simple manner. Changes are compared with a reference sample stored under identical conditions as the infected nuts. The plot allows, at a glance, the recognition of individual landmarks specific to a sample group as well as common features of the spectra. Each sample reveals an individual signal pattern. The plot facilitates the recognition of signals that belong to biological relevant metabolites. Betaine and five signals were identified that specifically changed upon mold infestation. Graphical abstract.

Determination of microbiological contamination, antibacterial and antioxidant activities of natural plant hazelnut (Corylus avellana L.) pollen.

The aim of our study is to determine microbial contamination, antibacterial and antioxidant activities of 14 pollen samples of Corylus avellana collected from different locations in Slovakia. Microbiological analysis was carried out in two steps: microbiological assays and studies of antibacterial activity of pollen extracts. The antimicrobial properties of pollen extracts were carried out with the disc-diffusion method. Methanol (70%), ethanol (70%) and distilled water were used for pollen extracts. Five strains of bacteria such as gram-negative (Salmonella enterica subsp. enterica CCM 3807, Escherichia coli CCM 2024, and Yersinia enterocolitica CCM 5671) and gram-positive (Staphylococcus aureus CCM 2461 and Bacillus thuringiensis CCM 19T) were tested. Antioxidant activity of pollen extracts was determined by the DPPH method. Bacterial analysis includes the determination of the total bacterial count ranged from 4.08 to 4.61 log CFU g-1, mesophilic aerobic bacteria ranged from 3.40 to 4.89 log CFU g-1, mesophilic anaerobic bacteria ranged from 3.20 to 4.52 log CFU g-1, coliform bacteria ranged from 3.30 to 4.55 log CFU g-1, yeasts and filamentous fungi ranged from 3.00 to 3.56 log CFU g-1. Microscopic filamentous fungi Aspergillus spp., Alternaria spp., Penicillium spp., Cladosporium spp., Rhizopus spp., and Paecylomyces spp. were isolated from hazelnut pollen. Yersinia enterocolitica was the most sensitive strain among ethanolic and methanolic pollen hazelnut extracts. Staphylococcus aureus was the most sensitive strain against aqueous hazelnut pollen extracts. We determined the following sensitivity against ethanol pollen extracts respectively: Yersinia enterocolitica > Salmonella enterica > Staphylococcus aureus > Bacillus thuringiensis > Escherichia coli. Methanol pollen extracts had shown following sensitivity: Yersinia enterocolitica > Salmonella enterica > Escherichia coli > Staphylococcus aureus > Bacillus thuringiensis. Aqueous extracts had shown the following sensitivity: Staphylococcus aureus > Salmonella enterica > Escherichia coli > Bacillus thuringiensis > Yersinia enterocolitica. Hazelnut pollen extracts have over 82% antioxidant capacity in samples from non-urban zones. An elevated level of antioxidant potential in the pollen is determined by its biological properties conditioned by biologically active substances. DPPH method allowed characterizing pollen as a source of antioxidants.

Fungi Associated with and Influence of Moisture on Development of Kernel Mold of Hazelnut.

Kernel mold on hazelnuts is defined by the United States Department of Agriculture, U.S.A., as any visible fungal growth either on the outside or inside of the kernel. Only one yeast and one filamentous fungus have been associated with kernel mold of hazelnut in Oregon. In this report, fungi were isolated from kernels with mold and identified using morphological and molecular characters. Penicillium spp. were isolated most often from kernels with mold, but species of Aspergillus and Cladosporium and Diaporthe rudis were also frequently isolated. Additional fungi from three other genera were also isolated. All of the same fungi were also isolated from symptomless kernels. Eremothecium coryli or Ramularia sp. previously associated with kernel defects in Oregon were not found associated with symptoms of kernel mold. Incidence of mold was the highest when nuts were incubated in moist chambers on wet, nonautoclaved orchard soil and was significantly higher than kernel mold found in nuts incubated on either air-dried soil or wet, autoclaved soil. Preventing hazelnuts from coming in contact with wet soil in the field using elevated wire screens resulted in significantly less mold development in two out of three years evaluated. Nuts on screens had a greater chance to dry out between rainstorms as measured by significantly lower nut moisture levels at harvest.

New synergistic co-culture of Corylus avellana cells and Epicoccum nigrum for paclitaxel production.

Paclitaxel is a main impressive chemotherapeutic agent with unique mode of action and broad-spectrum activity against cancers. Hazel (Corylus avellana) is a paclitaxel-producing species through bioprospection. Endophytic fungi have significant roles in plant paclitaxel production. This study evaluated the effect of co-culture of C. avellana cells and paclitaxel-producing endophytic fungus, Epicoccum nigrum strain YEF2 and also the effect of elicitors derived from this fungal strain on paclitaxel production. The results clearly revealed that co-culture of C. avellana cells and E. nigrum was more effective than elicitation of C. avellana cells by only cell extract or culture filtrate of this fungal strain. Co-culture of C. avellana cells and E. nigrum surpassed monocultures in terms of paclitaxel production designating their synergistic interaction potential. Fungal inoculum amount, co-culture establishment time and co-culture period were important factors for achieving the maximum production of paclitaxel in this co-culture system. The highest total yield of paclitaxel (404.5 µg L-1) was produced in co-culture established on 13th day using 3.2% (v/v) of E. nigrum mycelium suspension, which was about 5.5 and 136.6 times that in control cultures of C. avellana cells and E. nigrum, respectively. This is the first report on positive effect of co-culture of paclitaxel-producing endophytic fungus and non-host plant cells for enhancing paclitaxel production.

Elicitors Derived from Hazel (Corylus avellana L.) Cell Suspension Culture Enhance Growth and Paclitaxel Production of Epicoccum nigrum.

The microbial fermentation is considered as the potential source for large-scale production of paclitaxel. Since co-cultivation/mixed fermentation strategy has been reported as a yield enhancement strategy for paclitaxel production, investigation of fungal endophyte response to plant culture medium, plant cell extract (CE) and medium filtrate (MF) of plant cell suspension culture in terms of growth and paclitaxel production is interesting. In this study, 35 endophytic fungi were isolated from Taxus baccata and Corylus avellana grown in Iran. The analysis of high-performance liquid chromatography and mass spectrometry showed that one isolate (YEF2) produced paclitaxel. The isolate YEF2 was identified as Epicoccum nigrum by sequencing of ITS1-5.8S-ITS2 rDNA region and actin gene. YEF2 was slow-growing in Murashige and Skoog medium, but the synergistic interaction of gibberellic acid (GA3) and CE of C. avellana enhanced the growth of YEF2. The highest total yield of paclitaxel (314.7 µg/l; 11.5-folds) of E. nigrum strain YEF2 was obtained by using 28% (v/v) filter sterilized CE of C. avellana and 2 µg ml-1 GA3 that was significantly higher than the control. In this study, the effects of the plant cell extract on growth and paclitaxel production of paclitaxel producing endophytic fungus were studied for the first time.

Spirosoma pollinicola sp. nov., isolated from pollen of common hazel (Corylus avellana L.).

A Gram-negative bacterium, strain HA7T, was isolated from the microhabitat of common hazel (Corylus avellana L.) pollen. HA7T was found to be an aerobic, rod-shaped, catalase-positive, oxidase-negative bacterium with an optimum growth temperature of 25 °C and pH of 7. The nearly complete 16S rRNA gene sequence of HA7T strain showed the closest similarities to Spirosoma linguale DSM 74T (97.4 %) and Spirosoma fluviale DSM 29961T (97.43 %). The major fatty acids (>5 %) were C16 : 1ω5c, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), iso-C15 : 0 and iso-C17 : 0 3-OH. The major polar lipids were an unidentified aminophospholipid and phosphatidylethanolamine. The major respiratory quinone detected was menaquinone MK-7 (95 %). The draft genome sequence included 8 794 837 bases, which contained 3665 predicted coding sequences and had a G+C content of 47.9 mol%. The genome-based comparison between HA7T and S. linguale DSM 74T and S. fluviale DSM 29961T with pairwise average nucleotide identity indicated a clear distinction, between 76.2-76.3 %. Moreover, the digital DNA-DNA relatedness of HA7T to these strains was 26.5 and 25.1 %. Based on the differential genotypic, phenotypic and chemotaxonomic properties to closely related type strains, strain HA7T ought to be assigned with the status of a new species, for which the name Spirosomapollinicola sp. nov. is proposed. The type strain is HA7T (DSM 105799T=LMG 30282T).