Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins

In the pharmaceutical industry, improving the early detection of drug-induced hepatotoxicity is essential as it is one of the most important reasons for attrition of candidate drugs during the later stages of drug development. The first objective of this study was to better characterize different cellular models (i.e., HepG2, HepaRG cells, and fresh primary human hepatocytes) at the gene expression level and analyze their metabolic cytochrome P450 capabilities. The cellular models were exposed to three different CYP450 inducers; beta-naphthoflavone (BNF), phenobarbital (PB), and rifampicin (RIF). HepG2 cells responded very weakly to the different inducers at the gene expression level, and this translated generally into low CYP450 activities in the induced cells compared with the control cells. On the contrary, HepaRG cells and the three human donors were inducible after exposure to BNF, PB, and RIF according to gene expression responses and CYP450 activities. Consequently, HepaRG cells could be used in screening as a substitute and/or in complement to primary hepatocytes for CYP induction studies. The second objective was to investigate the predictivity of the different cellular models to detect hepatotoxins (16 hepatotoxic and 5 nonhepatotoxic compounds). Specificity was 100% with the different cellular models tested. Cryopreserved human hepatocytes gave the highest sensitivity, ranging from 31% to 44% (depending on the donor), followed by lower sensitivity (13%) for HepaRG and HepG2 cells (6.3%). Overall, none of the models under study gave desirable sensitivities (80–100%). Consequently, a high metabolic capacity and CYP inducibility in cell lines does not necessarily correlate with a high sensitivity for the detection of hepatotoxic drugs. Further investigations are necessary to compare different cellular models and determine those that are best suited for the detection of hepatotoxic compounds

Identification of Arthrinium marii as causal agent of olive tree dieback in Apulia (southern Italy)

Olive (Olea europaea L. var. sativa) is one of the most economically important tree crops grown in the Mediterranean basin. Arthrinium Kunze ex Fr. (teleomorph: Apiospora Sacc.) is a widespread fungal genus, and Arthrinium marii Larrondo & Calvo is a ubiquitous species, found in algae, soil, plants, and agricultural communities. A. marii was isolated from olive trees showing dieback from orchards located in Andria and in Fasano, Brindisi (Apulia, southern Italy) and identified based on morphological features and molecular analysis of four genomic regions (ITS, TUB2, TEF1, and LSU). Two-year-old olive plants artificially inoculated with three representative A. marii isolates showed complete dieback within 6 months, and the fungus was reisolated, satisfying Koch's postulates. This is the first report of A. marii causing dieback on olive trees that could represent an important threat for olive cultivation

Characterization of Monilinia spp. Populations on Stone Fruits in South Italy

Monilinia spp. are responsible for brown rot decay of stone and pome fruit in the field as well as in postharvest. Monilinia laxa and M. fructigena are considered indigenous to Europe, while M. fructicola is a quarantine pathogen in the European and Mediterranean Plant Protection Organization area included in the A2 List. In Italy, it was first reported in 2009 in Piedmont (northern Italy) and rapidly spread to central Italy. We carried out a monitoring program on the occurrence of Monilinia spp. in southern Italy and a comparative characterization of the three main fungal pathogens. Molecular assays based on direct polymerase chain reaction (PCR) and real-time quantitative PCR for molecular identification of Monilinia spp. from rotted fruit were set up, validated, and applied in a monitoring program. Of the tested 519 isolates from 26 orchards, 388 (74.8%) were identified as M. fructicola, 118 (22.7%) as M. laxa, 10 (1.9%) as M. fructigena, and 3 (0.6%) were M. polystroma. M. fructicola colonies grew faster and had a higher optimal temperature for growth (26°C) than M. laxa (23°C) and M. fructigena (20°C). No relevant difference in virulence could be observed on artificially inoculated apricot, cherry, and peach fruit. The fungal species showed different responses to fungicides, because M. fructicola was more sensitive than M. laxa, especially to cyflufenamid, and M. fructigena revealed a lower sensitivity to succinate dehydrogenase inhibitors (boscalid, fluopyram, and fluxapyroxad) and quinone outside inhibitors (mandestrobin). In summary, the two species M. fructicola and M. polystroma were first detected in southern Italy where M. fructicola has largely displaced the two indigenous pathogens M. laxa and M. fructigena; the relative proportions of the three pathogens in orchards should be considered when defining the management of brown rot of stone fruit due to differences in their responses to fungicides

RNA-Seq reveals OTA-related gene transcriptional changes in Aspergillus carbonarius

Ochratoxin A (OTA) is a mycotoxin harmful for animals and humans. Aspergillus carbonarius is the main responsible for OTA contamination of grapes and derived products. Gene transcriptional profiling of 4 A. carbonarius strains was carried out by RNA-Seq analysis to study transcriptome changes associated with OTA production. By comparing OTA inducing (OTAI) vs. non-inducing (OTAN) cultural conditions, a total of 3,705 differentially expressed genes (DEGs) (fold change > |2| and FDR ≤ 0.05) were identified. Several genes involved in primary metabolic processes, with particular regard to carbohydrate and amino acid metabolisms, secondary metabolic processes, transport, response to stress and sporulation were up-regulated by OTAI conditions at all the analysed sampling times (4, 6 and 8 DAI) or starting from 6 DAI. Highly up-regulated DEGs encoding enzymes involved in biosynthesis of secondary metabolites, oxidoreductases, transporters and transcription factors were examined for their potential involvement in OTA biosynthesis and related metabolic pathways. Differential expression of genes encoding polyketide synthases (pks), non-ribosomal peptide synthetases (nrps) and chloroperoxidase (cpo) was validated by RT-qPCR. Among clusters of co-regulated genes involved in SM biosynthesis, one putative OTA-gene cluster, including both pks and nrps genes, was detected in the A. carbonarius genome

First Report of Bacterial Spot caused by Xanthomonas arboricola pv. pruni on Almond in Italy

From July to September 2018, symptoms recalling those of bacterial infections were observed on different Prunus dulcis cultivars in several orchards of the Bari and Foggia provinces (southern Italy). Sunken and corked lesions oozing abundant gum occurred on the mesocarp of about 70 to 80% of the fruits, and dark circular spots were observed on the endocarp. Small, angular water-soaked spots surrounded by chlorotic tissue were initially observed along leaf midribs and on margins, turning light brown. The necrotic area was shed, giving rise to “shot-hole.” Sometimes the spots coalesced, resulting in large areas of necrotic foliar tissue. Isolations from diseased leaves and fruits were carried out on yeast extract–dextrose–calcium carbonate agar plates on which, after 4 days at 27°C, convex, smooth, mucoid, yellow, and glistening Xanthomonas-like colonies were observed. Three representative isolates were characterized as gram negative, oxidase negative, and catalase positive and able to hydrolyze esculin, gelatin, and Tween 80 but not starch (Schaad et al. 2001). Furthermore, these isolates utilized citrate, glycerol, and propionate, were hypersensitive in tobacco, digested casein, produced hydrogen sulfide from cysteine, and exhibited alkalinity from litmus milk but not indol, in accordance with the reactions of the reference strain NCPPB 416T of Xanthomonas arboricola pv. pruni (Xap). The identity of the isolates was confirmed by polymerase chain reaction (PCR) assays, sequencing, and pathogenicity tests. Genomic DNA, extracted from 24-h-old Luria-Bertani broth cultures of the representative bacterial isolates Xap-53, Xap-54, and Xap-55 and the reference Xap strain NCPPB 416T was successfully amplified in duplex-PCR with Xap-specific primers (XarbQ-F/XarbQ-R and XapY17-F/XapY17-R) (Pothier et al. 2011). Furthermore, the partial 16S rDNA genomic region was amplified using the primers fD1/rD1 (Weisburg et al. 1991) and custom sequenced (Genewiz, Takeley, U.K.). The sequences had 100% identity (e-value = 0; coverage 100%) with those of the same genomic region of other Xap strains, including the reference strain NCPPB 416 (GenBank accession nos. MK156160, DiSSPA_Xap_53; MK156161, DiSSPA_Xap_54; MK156162, DiSSPA _Xap_55; and MK156163, NCPPB 416). Pathogenicity was confirmed by infiltrating 107 CFU/ml of a bacterial suspension on detached leaves and leaves of 1-year-old potted plants of the almond cultivar Genco (Randhawa and Civerolo 1985). Control leaves were infiltrated with sterile distilled water. All leaves inoculated with the bacterial isolates and the reference strain developed confluent water-soaking spots 7 days postinoculation and incubation at 25°C and 16:8-h photoperiod. No symptoms were observed on controls. Bacteria reisolated from symptomatic leaf tissues exhibited the same morphological, biochemical, and molecular characteristics of the inoculated strains. In Europe, Xap was previously reported on almond in Montenegro (Panić et al. 1998) and Spain (Palacio-Bielsa et al. 2010). Although in Italy Xap has been considered endemic since the 1970s (Battilani et al. 1999), to our knowledge, this is the first report on almond. Xap is one of the most important diseases of stone fruits included in the EPPO A2 list of pests recommended for regulation for European Union member countries. Almond growing is expanding in Italy, and new technologies in crop management are being adopted. In this new scenario, together with climate changes, the pathogen could have a significant impact on almond production

First Report of Erysiphe sp. as a Causal Agent of Powdery Mildew on Punica granatum in Italy

Punica granatum L. (pomegranate), belonging to the Punicaceae family, is a plant native to the Middle East. Its fruit has gained widespread popularity as a functional and nutraceutical food and hence, pomegranate growing has noticeably increased in southern Italy from 43 ha in 2010 to 168 ha in 2015 (http://agri.istat.it/). In June 2015, a severe outbreak of powdery mildew was observed on 70% of 450,000 2-year-old pomegranate plants cvs. Wonderful One and Ako in a nursery located in Lecce Province, Italy. White, dense plaques of mycelium and conidia covering 30 to 40% of the young leaves were observed. At later stages of disease development, leaves turned yellow and abscised. No chasmothecia were detected. The conidiophore foot cell was cylindrical and the appressorium lobed. Microscopic observations of 300 conidia from each of 10 plant samples showed that they were hyaline, ellipsoid to cylindrical, measuring 28.1 to 35.7 × 12.9 to 14.4 μm (average 32.8 × 13.4 μm), fibrosin bodies were absent, and the germ tube was subterminal. ITS genomic regions were sequenced for molecular identification. DNA was extracted from conidia and mycelium by using InstaGene Matrix (Bio-Rad Laboratories, Hercules, CA), amplified by PCR using the universal ITS5/ITS4 primers, and the amplicon was sequenced by external service (Macrogen, Seoul, South Korea). BLASTn analysis of the ITS sequence (563 bp) showed a high homology (identity: 99%; e-value: 0.0; coverage: 99%) with species of the Erysiphe aquilegiae clade (i.e., E. aquilegiae [EU047570.1], E. sedi [JX173288.1], Pseudoidium hortensiae [JQ669944.1], and P. neolycopersici [GU358451.1]) (Takamatsu et al. 2015). Artificial inoculations were made by gently pressing naturally diseased leaves onto 20 young fully expanded leaves of 10 healthy potted plants cv. Wonderful One grown under glasshouse conditions at 25 ± 2°C. Five noninoculated plants served as controls. Typical powdery mildew symptoms developed within 7 to 10 days only on inoculated leaves. Therefore, on the basis of morphological features, the pathogen was tentatively identified as Erysiphe sp. and, according to the BLASTn results, it appears to belong to the still unresolved E. aquilegiae clade. The ITS sequence was deposited in GenBank under accession number KU060880. E. punicae T.M. Achundov, whose gene sequences are not available, was reported as the causal agent of powdery mildew on pomegranate in Azerbaijan (Akhundov 1987), Iran (Khodaparast and Abbasi 2009), and Montenegro; anamorphs of powdery mildews on pomegranate were also recorded from Ethiopia, Greece, India, Iraq, Ukraine, and Crimea but it is unclear if they belong to E. punicae (Braun and Cook 2012). This is the first report of powdery mildew on pomegranate in Italy. Due to the poor availability of fungicides allowed on the crop, improved disease management methods are needed to prevent heavy yield losses. References: Akhundov, T. M. 1987. Nov. Sist. Niz. Rast. 24:95. Braun, U., and Cook, R.T.A. 2012. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No. 11. CBS, Utrecht, The Netherlands. Khodaparast, S. A., and Abbasi, M. 2009. Mycotaxon 108:213. 10.5248/108.213. Takamatsu, S., et al. 2015. Mycologia 107:475. 10.3852/15-00

First report of Pseudomonas grapevine bunch rot caused by Pseudomonas syringae pv syringae

Pseudomonas syringae pv. syringae, a Gammaproteobacterium belonging to genomospecies 2 within the P. syringae complex, is distributed worldwide, and it is responsible for bacterial canker on >100 different hosts, including the grapevine. P. syringae pv. syringae induces necrotic lesions in the leaf blades, veins, petioles, shoots, rachis, and tendrils on grapevine cultivars in different areas. P. syringae pv. syringae has been associated with severe economic losses in different grape cultivars in Australia, where it causes inflorescence rot. In midsummer to late summer 2017, symptoms of berry rots differing from those caused by the common berry rots agents were observed in different cultivar Red Globe vineyards of Apulia (southern Italy). As proven by fulfillment of Koch's postulates, these symptoms were caused by a bacterium that, according to the results of biochemical, physiological, nutritional, antimicrobial activity, and pathogenicity tests and sequencing of 16S ribosomal DNA, gyrB, rpoB, and rpoD genes, was identified as P. syringae pv. syringae. This is the first report of Pseudomonas grapevine bunch rot

The Mycovirome in a Worldwide Collection of the Brown Rot Fungus Monilinia fructicola

The fungus Monilinia fructicola is responsible for brown rot on stone and pome fruit and causes heavy yield losses both pre- and post-harvest. Several mycoviruses are known to infect fungal plant pathogens. In this study, a metagenomic approach was applied to obtain a comprehensive characterization of the mycovirome in a worldwide collection of 58 M. fructicola strains. Deep sequencing of double-stranded (ds)RNA extracts revealed a great abundance and variety of mycoviruses. A total of 32 phylogenetically distinct positive-sense (+) single-stranded (ss)RNA viruses were identified. They included twelve mitoviruses, one in the proposed family Splipalmiviridae, and twelve botourmiaviruses (phylum Lenarviricota), eleven of which were novel viral species; two hypoviruses, three in the proposed family Fusariviridae, and one barnavirus (phylum Pisuviricota); as well as one novel beny-like virus (phylum Kitrinoviricota), the first one identified in Ascomycetes. A partial sequence of a new putative ssDNA mycovirus related to viruses within the Parvoviridae family was detected in a M. fructicola isolate from Serbia. The availability of genomic sequences of mycoviruses will serve as a solid basis for further research aimed at deepening the knowledge on virus–host and virus–virus interactions and to explore their potential as biocontrol agents against brown rot disease

De novo assembly and comparative transcriptome analysis of Monilinia fructicola, Monilinia laxa and Monilinia fructigena, the causal agents of brown rot on stone fruits

Background: Brown rots are important fungal diseases of stone and pome fruits. They are caused by several Monilinia species but M. fructicola, M. laxa and M. fructigena are the most common all over the world. Although they have been intensively studied, the availability of genomic and transcriptomic data in public databases is still scant. We sequenced, assembled and annotated the transcriptomes of the three pathogens using mRNA from germinating conidia and actively growing mycelia of two isolates of opposite mating types per each species for comparative transcriptome analyses. Results: Illumina sequencing was used to generate about 70 million of paired-end reads per species, that were de novo assembled in 33,861 contigs for M. fructicola, 31,103 for M. laxa and 28,890 for M. fructigena. Approximately, 50% of the assembled contigs had significant hits when blasted against the NCBI non-redundant protein database and top-hits results were represented by Botrytis cinerea, Sclerotinia sclerotiorum and Sclerotinia borealis proteins. More than 90% of the obtained sequences were complete, the percentage of duplications was always less than 14% and fragmented and missing transcripts less than 5%. Orthologous transcripts were identified by tBLASTn analysis using the B. cinerea proteome as reference. Comparative transcriptome analyses revealed 65 transcripts over-expressed (FC ≥ 8 and FDR ≤ 0.05) or unique in M. fructicola, 30 in M. laxa and 31 in M. fructigena. Transcripts were involved in processes affecting fungal development, diversity and host-pathogen interactions, such as plant cell wall-degrading and detoxifying enzymes, zinc finger transcription factors, MFS transporters, cell surface proteins, key enzymes in biosynthesis and metabolism of antibiotics and toxins, and transposable elements. Conclusions: This is the first large-scale reconstruction and annotation of the complete transcriptomes of M. fructicola, M. laxa and M. fructigena and the first comparative transcriptome analysis among the three pathogens revealing differentially expressed genes with potential important roles in metabolic and physiological processes related to fungal morphogenesis and development, diversity and pathogenesis which need further investigations. We believe that the data obtained represent a cornerstone for research aimed at improving knowledge on the population biology, physiology and plant-pathogen interactions of these important phytopathogenic fungi