Triazole resistance in Aspergillus fumigatus isolated from a tomato production environment exposed to propiconazole.

The emergence of azole-resistant Aspergillus fumigatus (ARAf) across the world is an important public health concern. We sought to determine if propiconazole, a demethylase inhibitor (DMI) fungicide, exerted a selective pressure for ARAf in a tomato production environment following multiple exposures to the fungicide. A tomato field trial was established in 2019 and propiconazole was applied weekly until harvest. Soil, leaf, and fruit (when present) samples were collected at baseline and after each propiconazole application. A. fumigatus isolates (n, 178) were recovered and 173 were tested for susceptibility to itraconazole, posaconazole, voriconazole, and propiconazole in accordance with CLSI M38 guidelines. All the isolates were susceptible to medical triazoles and the propiconazole MIC ranged from 0.25 to 8 mg/L. A linear regression model was fitted that showed no longitudinal increment in the log2-fold azole MIC of the isolates collected after each propiconazole exposure compared to the baseline isolates. AsperGenius real-time multiplex assay ruled out TR34/L98H and TR46/Y121F/T289A cyp51A resistance markers in these isolates. Sequencing of a subset of isolates (n, 46) demonstrated widespread presence of F46Y/M172V/E427K and F46Y/M172V/N248T/D255E/E427K cyp51A mutations previously associated with reduced susceptibility to triazoles. IMPORTANCE: The agricultural use of azole fungicides to control plant diseases has been implicated as a major contributor to ARAf infections in humans. Our study did not reveal imposition of selection pressure for ARAf in a vegetable production system. However, more surveillance studies for ARAf in food crop production and other environments are warranted in understanding this public and One Health issue.

First report of Colletotrichum scovillei causing anthracnose fruit rot of pepper in Ohio.

In August 2019, the Ohio State University Vegetable Pathology laboratory received multiple bell and banana pepper fruits (Capsicum annuum, cvs. unknown) from Columbiana County, Ohio. The grower reported a disease incidence of 100% and severity of 70% in fruits across their pepper fields. Fruit lesions were brown, sunken, and covered with orange-colored sporulation. On banana peppers, the lesions mainly affected the blossom end of the fruits, while the lesions were distributed randomly on bell pepper fruits. Pieces of diseased tissue were cut from the fruit and surface sterilized in 0.5-0.6 % sodium hypochlorite, rinsed in sterile water, blotted dry, and placed on potato dextrose agar. All of the fungal cultures recovered were cottony, pale gray-green with shades of orange on the underside of the mycelial mat. Two representative isolates, SM209-19 (bell pepper) and SM210-19 (banana pepper), were grown on oatmeal agar to induce sporulation. Pink-orange concentric rings containing acervuli and conidia were present on the oatmeal agar plates after one week of growth at 22◦C (12-h dark/light). Conidia (n=29) were hyaline, aseptate, cylindrical in shape, and had an average length of 10.5 µm (std. dev. = 1.3 µm) and width of 4.1 µm (std. dev. = 0.6 µm) (Fig.1). DNA was extracted from both isolates using a DNeasy Plant Kit (Qiagen Inc, Germantown, MD), and partial sequences of the internal transcribed spacer (ITS) region, -tubulin 2 gene (TUB2), and glyceraldehyde-3-phosphate dehydrogenase gene (GDPH) were amplified by PCR with the following primers: ITS4/ITS5 (White et al. 1990), Bt1a/Bt1b (Glass et al. 1995), and GDF1/GDR1 (Guerber et al. 2003), and squenced. The ITS region of both isolates SM209-19 and SM210-19 (PP280815 and PP280816, respectively) showed 100% identity with C. scovillei (Cs) isolate LJTJ35 (KP748226). The partial sequences of GDPH, (PP320348, PP320349, respectively) showed 99% sequence identity with the Cs CBS 126528 (JQ948597) and 100% identity with Cs HP1 (MT826948) The partial sequences of TUB2 (PP472464 and PP472465, respectively) had 100% sequence similarity with the Cs HP1 and Cs CBS 126528 (MT826951, JQ949918 respectively). Pathogenicity was tested in a greenhouse experiment on blossoming bell pepper plants (cv. Carmen) by spraying 10 ml of 1 X 105 conidia/ml suspension onto flower blooms (nine plants per isolate). Control pepper plants were mock inoculated by spraying 10 mL of sterile deionized water. The plants were re-inoculated one week later. Brown, sunken lesions with orange sporulation developed on the fruits of inoculated plants 21 days after the initial inoculation (Fig. 2), while the mock-inoculated plants did not produce any symptoms. Culturing from symptomatic fruits on PDA, following the same method described above, produced fungal colonies with the same morphological traits previously described. C. scovillei causing anthracnose on pepper has been described in the US (Toporek et al. 2021), Brazil (Caires et al. 2014), China (Zhao et al. 2016), and different South Asia Countries (Khalimi et al. 2019). Open-field peppers are produced in Ohio on more than 5,400 acres, with a value of more than $53 million, with anthracnose being one of the most severe fungal diseases reducing yield. This newly reported Colletotichum species could represent a further threat for this crop. Further studies evaluating fungicide sensitivity and efficacy against this pathogen will be of crucial importance for disease management.

First Report of Diaporthe fruit rot and stem canker of hydroponic cucumber caused by Diaporthe sp. in the United States.

Multiple Diaporthe spp. cause root and fruit rots or stem lesions on Cucumis spp.: D. cucurbitae, D. melonis, D. longicolla (syn. D. eres), D. pterocarpi, D. sclerotioides, D. sojae, and D. ueckerae (Broge et al. 2020; Fukada et al. 2018; Udayanga et al. 2012, 2015). From May-August 2021, cucumbers (Cucumis sativus) 'Katrina' and 'Alcazar' were grown in a 24-plant, commercial Bato bucket system with rockwool blocks on a perlite substrate in a research greenhouse in Wooster, Ohio. At maturity, plants collapsed rapidly from stem lesions without foliar chlorosis (25% of 'Katrina' and 17% of 'Alcazar'). Lesions were 7.5 to 15 cm in length, tan to golden-brown with black pycnidia and located 5 to 15 cm above the crown. Stems shredded easily with vascular discoloration around the lesion. Two identical fungal strains were isolated on ½ acidified potato dextrose agar (APDA) following surface disinfestation with 0.6% sodium hypochlorite for 30 s and sterile water rinse. Fungal cultures were floccose, white to tan mycelia with pycnidia. Oblong, elliptical, biguttulate, aseptate alpha conidia were observed with mean dimensions: 8.0 µm (5.2-9.8 µm) by 3.1 µm (2.5-3.8 µm) on ½ APDA and 9.8 µm (6.6-12.4 µm) by 3.0 µm (1.9-5.3 µm) on petioles. On prune extract agar, beta conidia mean dimensions were: 19.7 µm (12.0-27.7 µm) by 1.2 µm (0.8-1.8 µm). Fungal DNA was amplified and sequenced bidirectionally with ITS (ITS4/ITS5), CAL (CAL228F/737R), HIS (CYLH3F/H3-1B), TEF1 (EF1-728f/EF1-986R), and TUB2 (Bt1a/Bt1b) primers (Carbone and Kohn 1999; Glass and Donaldson 1995) (GenBank: OP265712-13, OP288460-65, OQ418506-07). Based on a maximum likelihood phylogenetic tree of concatenated genes, this novel Diaporthe sp., most closely related to D. stewartii, has not been reported on Cucumis spp. Strains were deposited in the USDA-ARS Culture Collection (NRRL# 64461-62). Koch's postulates were conducted in a greenhouse with mean day temperature of 25°C and 12 hr supplemental lighting. One-month old cucumbers 'Katrina,' grown in rockwool cubes (5 plants per isolate) and potting mix (6 plants per isolate), were inoculated with a one-week-old culture of either strain. The second true leaf was cut and a pipette tip containing an inoculated plug of ½ APDA was placed on the remaining petiole (Mathew et al. 2018). Non-inoculated ½ APDA was used for controls. Plants were tarped for 24 hours to increase humidity and pipette tips removed after one week. After two weeks, petioles were shrunken, tan to golden brown with pycnidia. After 3-4 weeks, stem lesions matching those above were observed on inoculated plants, and plants collapsed. For fruit rot, three Beit Alpha cucumbers were rinsed with tap water, dried, a 5 mm plug was removed from the fruit and replaced with a 5 mm plug of one-week-old fungus on ½ APDA. After 3 days, fruits were water soaked and soft. For root rot, two plates of one-week-old cultures were macerated in 500 mL of sterile water and mixed with 1500 mL of vermiculite. Two seeds of cucumber 'Katrina' were planted into three reps of each isolate and control. All control seeds germinated, but all inoculated seeds experienced pre- or post-emergence damping off. No symptoms were ever observed on any controls. Fungi were isolated from all inoculated tissues as described above. Based on morphology, Diaporthe sp. was isolated from all inoculated plants but never from controls. This Diaporthe sp. may be a new constraint to hydroponic cucumber production, but incidence needs to be determined globally.

A Quantitative PCR Method to Detect the Tomato Corky Root Rot Pathogens, Pseudopyrenochaeta lycopersici and P. terrestris.

Corky root rot is an important disease in tomato production systems and is caused by Pseudopyrenochaeta terrestris and P. lycopersici (formerly Pyrenochaeta lycopersici Types 1 and 2, respectively). The corky root rot pathogens are slow growing and difficult to isolate and quantify in soil and plant tissue. A multiplex hydrolysis probe-based qPCR assay was designed to allow for simultaneous detection and quantification of P. lycopersici and P. terrestris with a competitive internal control to indicate if qPCR inhibitors are present. Single species and multiplex assays for Pseudopyrenochaeta spp. detected DNA levels above 0.013 pg of DNA per reaction. These highly specific assays had no nontarget amplification of other fungal and oomycete pathogens or rhizosphere-associated fungi of tomatoes that were tested. This assay can be used to quantify Pseudopyrenochaeta populations in roots and soils in tomato production systems to better determine the impacts of disease management strategies on Pseudopyrenochaeta spp. and provides a tool to study the biology of Pseudopyrenochaeta spp.

Discovery of Novel Small Molecule Growth Inhibitors to Manage Pseudomonas Leaf Spot Disease on Peppers (Capsicum sp.).

Pseudomonas leaf spot (PLS) disease in peppers caused by Pseudomonas syringae pv. syringae (Pss) is an emerging seedborne phytopathogen. Pss infection can severely reduce the marketable yield of peppers in favorable environmental conditions and cause significant economic losses. The intensive use of copper-sulfate and streptomycin-sulfate to control PLS and other bacterial diseases is associated with antimicrobial-resistant Pss strains, making these control methods less effective. So, there is an urgent need to develop novel antimicrobials effective against Pss in peppers. Several studies, including those done in our laboratory, have shown that small molecule (SM) antimicrobials are ideal candidates as they can be effective against multidrug resistant bacteria. Therefore, our study aims to identify novel SM growth inhibitors of Pss, assess their safety, and evaluate their efficacy on Pss-infected pepper seeds and seedlings. Using high-throughput screening, we identified 10 SMs (PC1 to PC10) that inhibited the growth of Pss strains at 200 µM or lower concentrations. These SMs were effective against both copper- and streptomycin-resistant as well as biofilm-embedded Pss. These SMs were effective against other plant pathogens (n = 22) at low concentrations (<200 µM) and had no impact on beneficial phytobacteria (n = 12). Furthermore, these SMs showed better or equivalent antimicrobial activity against Pss in infested pepper seeds and inoculated seedlings compared with copper-sulfate (200 µM) and streptomycin (200 µg/ml). Additionally, none of the SMs were toxic to pepper tissues (seeds, seedlings, or fruits), human Caco-2 cells, and pollinator honeybees at 200 µM. Overall, the SMs identified in this study are promising alternative antimicrobials for managing PLS in pepper production.

First Report of Neopestalotiopsis Disease in Ohio caused by an emerging and novel species of Neopestalotiopsis on Strawberry.

In October 2021, strawberry (Fragaria x ananassa) plants (cv. Ruby June) that had dark brown lesions with a diffuse black halo and light brown center and / or dark brown V-shaped necrotic areas often starting from the edge of the leaves were observed in a commercial planting in Washington County. The grower reported 50% incidence in the field when the sample was first submitted and two weeks later reported 80% incidence. The morphology of conidia present on symptomatic leaf tissue was consistent with species of Neopestalotiopsis (Maharachchikumbura et al. 2014). The conidia were ellipsoid to fusiform, five-celled, with three light brown colored median cells and one hyaline apical and basal cell. The apical cells had two to four flexuous appendages and the basal cell had one non-flexuous appendage. Average (N=30) conidia length, not including the appendages, and width was 24.1 ± 2.7 and 6.5 ± 1.4 µm respectively. Two isolates (MLI267-21 and MLI268-21) were purified on potato dextrose agar, producing a dense white mycelial mat with undulate margins. The underside color of the mycelial mat was pinkish-orange. Conidiomata formed randomly in the colonies and extruded black gelatinous spores. To confirm the identity of these isolates the genome of MLI267-21 was sequenced using the NextSeq 2000 Illumina platform and Nextera DNA CD indexes (OSU Applied Microbiology Service Laboratory, Columbus, OH). Partial internal transcribed spacer (ITS) region, ß-tubulin (TUB), and translation elongation factor 1-alpha (TEF-1α) gene sequences (Accession numbers: OM649904, OM649905, and OM649906 respectively) were extracted from the MLI267-21 genome, concatenated, and aligned to published reference sequences. These same genes were amplified and sequenced from MLI268-21. Maximum likelihood phylogenetic analysis performed in IQ-TREE (Minh et al. 2020, Kalyaanamoorthy et al. 2017, Chernomor et al. 2016) with the aligned sequences revealed the clustering of MLI267-21 and MLI268-21 with seven other Neopestalotiopsis isolates, from strawberry (17-43L; Baggio et al. 2021) and pomegranate (GEV3426 to GEV3431; Xavier et al. 2021) leaves in Florida, which form a unique and emerging species group. The ITS, TUB, and TEF-1α sequences from both Ohio isolates were 100% similar to the same sequences from 17-43L and GEV3426 - GEV3431. Pathogenicity tests were performed using MLI267-21 by spray inoculating (~104 spore/ml) four-week-old 'Cabrillo' strawberry plants (n=4) and placing three drops (10µl each) of spore suspension (~104 spore/ml) on the calix area of detached fruit (n=4). Non-inoculated plants and fruit (n= 4 each) served as negative controls. The plants were covered with transparent plastic bags and maintained at 25 °C for 72 hours before the bags were removed (Baggio et al. 2021). Five days post-inoculation, dark brown circular spots on the leaves and petioles were observed on all four inoculated plants and acervuli were observed within the necrotic spots after an additional 72 hours in a moist chamber. Fruits were incubated in a moist chamber at 25 °C and after 72 hours orange-brown lesions formed on the fruit. After five days, fruit were mushy and covered with white mycelia, acervuli, and conidiomata. Neopestalotiopsis disease has been reported on strawberry in Florida (Baggio et al. 2021) and in several South American (Obregon et al. 2018, Hidrobo et al. 2021) and European (Chamorro et al. 2016, Gilardi et al. 2019) countries. The disease can cause rapid plant death when conditions are warm and wet. Research to investigate host susceptibility and to identify effective chemical and biological control has been initiated in Ohio to establish preventative management programs for commercial field operations.

Mechanistic Interrogation of Cell Transformation In Vitro: The Transformics Assay as an Exemplar of Oncotransformation.

The Transformics Assay is an in vitro test which combines the BALB/c 3T3 Cell Transformation Assay (CTA) with microarray transcriptomics. It has been shown to improve upon the mechanistic understanding of the CTA, helping to identify mechanisms of action leading to chemical-induced transformation thanks to RNA extractions in specific time points along the process of in vitro transformation. In this study, the lowest transforming concentration of the carcinogenic benzo(a)pyrene (B(a)P) has been tested in order to find molecular signatures of initial events relevant for oncotransformation. Application of Enrichment Analysis (Metacore) to the analyses of the results facilitated key biological interpretations. After 72 h of exposure, as a consequence of the molecular initiating event of aryl hydrocarbon receptor (AhR) activation, there is a cascade of cellular events and microenvironment modification, and the immune and inflammatory responses are the main processes involved in cell response. Furthermore, pathways and processes related to cell cycle regulation, cytoskeletal adhesion and remodeling processes, cell differentiation and transformation were observed.

Novel Small Molecule Growth Inhibitors of Xanthomonas spp. Causing Bacterial Spot of Tomato.

Bacterial spot (BS) of tomato, caused by Xanthomonas gardneri, X. perforans, X. vesicatoria, and X. euvesicatoria, is difficult to control because of the high prevalence of copper- and streptomycin-resistant strains and the lack of resistance cultivars and effective bactericides. The objective of this study was to identify novel growth inhibitors of BS-causing Xanthomonas (BS-X) species by using small molecules (SM; n = 4,182). Several SMs (X1, X2, X5, X9, X12, and X16) completely inhibited the growth of BS-X isolates (n = 68 X. gardneri, 55 X. perforans, 4 X. vesicatoria, and 32 X. euvesicatoria) at ≥12.5 µM by disrupting Xanthomonas cell integrity through weakening of the cell membrane and formation of pores. These SMs were also effective against biofilm-embedded, copper- and streptomycin-resistant Xanthomonas strains while having minimal impact on other plant pathogenic (n = 20) and beneficial bacteria (n = 12). Furthermore, these SMs displayed equivalent antimicrobial activity against BS-X in seeds and X. gardneri in seedlings compared with conventional control methods (copper sulfate and streptomycin) at similar concentrations while having no detectable toxicity to tomato tissues. SMs X2, X5, and X12 reduced X. gardneri, X. perforans, X. vesicatoria, and X. euvesicatoria populations in artificially infested seeds ≤3.4-log CFU/seed 1 day postinfection (dpi) compared with the infested untreated control (P ≤ 0.05). SMs X1, X2, X5, and X12 reduced disease severity ≤72% and engineered bioluminescent X. gardneri populations ≤3.0-log CFU/plant in infected seedlings at 7 dpi compared with the infected untreated control (P ≤ 0.05). Additional studies are needed to increase the applicability of these SMs for BS management in tomato production.

First report of Xanthomonas hortorum causing bacterial leaf spot of lavender (Lavandula × intermedia) in Ohio.

In July 2018, a sample of lavender var. Grosso (Lavandula × intermedia 'Grosso') from Miami County, OH was received by The Ohio State University Vegetable Pathology Laboratory in Wooster. Lavender plants were field-grown in sandy clay soil with plastic mulch under drip irrigation. Disease incidence ranged from 0 to 32% depending on variety. Leaves and stems showed dark necrotic lesions that varied from roughly circular (ca. 0.3 to 0.5 mm diameter) to large coalesced necrotic areas surrounded by a water-soaked halo. Bacterial streaming from lesions was observed microscopically. Leaf tissue pieces (~0.5 cm2) were surface sterilized in 70% ethanol for 30 seconds and rinsed in sterile deionized water. The tissue was sliced aseptically into smaller sections in 100 µl sterile water and the bacterial suspension was streaked on yeast dextrose calcium carbonate agar medium. Ten yellow Xanthomonas-like colonies were selected after 72 hours of incubation at 28ºC in the dark. Strains were gram negative, oxidase negative and caused hypersensitive reactions on Nicotiana benthamiana (L.). All strains were genotyped after whole-cell DNA extraction by BOX-PCR (Louws et al. 1999) and had the same banding profile. Four 8-wk-old lavender plants (Lavandula dentata and Lavandula × ginginsii 'Goodwin Creek Gray') were spray-inoculated with a 106 CFU/ml suspension of strain SM175-2018 in sterile water. Control plants were sprayed with sterile water. Plants were kept in plastic bags for the first 48 h at 28°C with a 14-h photoperiod. Water-soaked necrotic lesions appeared 14 days after inoculation with SM175-2018, whereas mock-inoculated plants did not show symptoms. Bacterial isolation from symptomatic leaf tissue was carried out as described above. The BOX-PCR profile of the re-isolated strain was identical to that of SM175-2018. Multilocus sequence analysis of the housekeeping genes fuyA, gyrB, and rpoD was performed (Accession numbers: MT764834 - MT764836). The resulting concatenated data set was used to perform a phylogenetic analysis using maximum likelihood criteria to evaluate relationships with closely related Xanthomonas spp. using published reference sequences (Young et al. 2008). SM175-2018 was assigned to the X. hortorum clade (Moriniere et al. 2020) with strong bootstrap support. The strain was subjected to whole genome analysis. Genomic DNA was extracted using a QIAGEN Genomic DNA buffer set with genomic-tip 100/G following manufacturer's protocol and sequenced using the iSeq-100 Illumina platform with the Nextera DNA Flex Library Prep protocol kit and Nextera DNA CD indexes. Average nucleotide identity (ANI) analysis was performed with the ANI-Matrix software Enveomics tool (Rodriguez-R and Konstantinidis 2016) using the sequenced genome (NCBI GenBank Biosample no. SAMN11831455) and those of other X. hortorum (Vauterin et al. 1995) bacteria (pvs. hederae, carotae, vitians). SM175-2018 shared a 96% ANI with other X. hortorum strains. X. hortorum is associated with bacterial leaf spot of carrot (Scott and Dung, 2020) and also reported on ornamental plants (Mirik et al. 2010, Oliver et al. 2012, Roberts and Parkinson 2014, Klass et al. 2019), however additional research is needed to establish the host specificity of lavender strains. To our knowledge this is the first report of X. hortorum causing bacterial leaf spot of lavender in Ohio. The disease may negatively impact the yield and quality of flowers used in production of lavender oils and essences.

Environmental pollution and COVID-19: the molecular terms and predominant disease outcomes of their sweetheart agreement.

As the Coronavirus situation (COVID-19) continues to evolve, many questions concerning the factors relating to the diffusion and severity of the disease remain unanswered.Whilst opinions regarding the weight of evidence for these risk factors, and the studies published so far are often inconclusive or offer contrasting results, the role of comorbidities in the risk of serious adverse outcomes in patients affected with COVID-19 appears to be evident since the outset. Hypertension, diabetes, and obesity are under discussion as important factors affecting the severity of disease. Air pollution has been considered to play a role in the diffusion of the virus, in the propagation of the contagion, in the severity of symptoms, and in the poor prognosis. Accumulating evidence supports the hypothesis that environmental particulate matter (PM) can trigger inflammatory responses at molecular, cellular, and organ levels, sustaining respiratory, cardiovascular, and dysmetabolic diseases.To better understand the intricate relationships among pre-existing conditions, PM, and viral infection, we examined the response at the molecular level of T47D human breast adenocarcinoma cells exposed to different fractions of PM. T47D cells express several receptors, including the aryl hydrocarbon receptor (AhR), and ACE2, the main - but not the only - receptor for SARS-CoV-2 entry.PM samples were collected in an urban background site located in the Northern area of the City of Bologna (Emilia-Romagna Region, Northern Italy) during winter 2013. T47D cells were exposed to organic or aqueous (inorganic) extracts at the final concentration of 8 m3 for a 4-hour duration. Both the concentration and the exposure time were chosen to resemble an average outdoor exposure. RNA was extracted from cells, purified and hybridised on 66k microarray slides from Agilent.The lists of differentially expressed genes in PM organic extracts were evaluated by using Metacore, and an enrichment analysis was performed to identify pathways maps, process networks, and disease by biomarkers altered after T47D treatment.The analysis of the modulated genes gave evidence for the involvement of PM in dysmetabolic diseases, including diabetes and obesity, and hypertension through the activation of the aryl hydrocarbon receptor (AhR) canonical pathway.On the basis of current knowledge, existing data, and exploratory experimental evidence, we tease out the likely molecular interplay that can ultimately tip the disease outcome into severity. Looking beyond ACE2, several additional key markers are identified. Disruption of these targets worsens pre-existing conditions and/or exacerbates the adverse effects induced by SARS-CoV-2 infection. Whilst appropriately designed, epidemiological studies are very much needed to investigate these associations based on our hypothesis of investigation, by reviewing recent experimental and epidemiological evidence, here we speculate and provide new insights on the possible role of environmental pollution in the exacerbation of effects by SARS-CoV-2 and other respiratory viruses. This work is intended to assist in the development of appropriate investigative approaches to protect public health.

The transformics assay: first steps for the development of an integrated approach to investigate the malignant cell transformation in vitro.

The development of alternative methods to animal testing is a priority in the context of regulatory toxicology. Carcinogenesis is a field where the demand for alternative methods is particularly high. The standard rodent carcinogenicity bioassay requires a large use of animals, high costs, prolonged duration and shows several limitations, which can affect the comprehension of the human relevance of animal carcinogenesis. The cell transformation assay (CTA) has long been debated as a possible in vitro test to study carcinogenesis. This assay provides an easily detectable endpoint of oncotransformation, which can be used to anchor the exposure to the acquisition of the malignant phenotype. However, the current protocols do not provide information on either molecular key events supporting the carcinogenesis process, nor the mechanism of action of the test chemicals. In order to improve the use of this assay in the integrated testing strategy for carcinogenesis, we developed the transformics method, which combines the CTA and transcriptomics, to highlight the molecular steps leading to in vitro malignant transformation. We studied 3-methylcholanthrene (3-MCA), a genotoxic chemical able to induce in vitro cell transformation, at both transforming and subtransforming concentrations in BALB/c 3T3 cells and evaluated the gene modulation at critical steps of the experimental protocol. The results gave evidence for the potential key role of the immune system and the possible involvement of the aryl hydrocarbon receptor (AhR) pathway as the initial steps of the in vitro transformation process induced by 3-MCA, suggesting that the initiating events are related to non-genotoxic mechanisms.

In vitro, in planta, and comparative genomic analyses of Pseudomonas syringae pv. syringae strains of pepper (Capsicum annuum var. annuum).

Pseudomonas syringae pv. syringae (Pss) is an emerging phytopathogen that causes Pseudomonas leaf spot (PLS) disease in pepper plants. Pss can cause serious economic damage to pepper production, yet very little is known about the virulence factors carried by Pss that cause disease in pepper seedlings. In this study, Pss strains isolated from pepper plants showing PLS symptoms in Ohio between 2013 and 2021 (n = 16) showed varying degrees of virulence (Pss populations and disease symptoms on leaves) on 6-week-old pepper seedlings. In vitro studies assessing growth in nutrient-limited conditions, biofilm production, and motility also showed varying degrees of virulence, but in vitro and in planta variation in virulence between Pss strains did not correlate. Comparative whole-genome sequencing studies identified notable virulence genes including 30 biofilm genes, 87 motility genes, and 106 secretion system genes. Additionally, a total of 27 antimicrobial resistance genes were found. A multivariate correlation analysis and Scoary analysis based on variation in gene content (n = 812 variable genes) and single nucleotide polymorphisms within virulence genes identified no significant correlations with disease severity, likely due to our limited sample size. In summary, our study explored the virulence and antimicrobial gene content of Pss in pepper seedlings as a first step toward understanding the virulence and pathogenicity of Pss in pepper seedlings. Further studies with additional pepper Pss strains will facilitate defining genes in Pss that correlate with its virulence in pepper seedlings, which can facilitate the development of effective measures to control Pss in pepper and other related P. syringae pathovars. IMPORTANCE: Pseudomonas leaf spot (PLS) caused by Pseudomonas syringae pv. syringae (Pss) causes significant losses to the pepper industry. Highly virulent Pss strains under optimal environmental conditions (cool-moderate temperatures, high moisture) can cause severe necrotic lesions on pepper leaves that consequently can decrease pepper yield if the disease persists. Hence, it is important to understand the virulence mechanisms of Pss to be able to effectively control PLS in peppers. In our study, in vitro, in planta, and whole-genome sequence analyses were conducted to better understand the virulence and pathogenicity characteristics of Pss strains in peppers. Our findings fill a knowledge gap regarding potential virulence and pathogenicity characteristics of Pss in peppers, including virulence and antimicrobial gene content. Our study helps pave a path to further identify the role of specific virulence genes in causing disease in peppers, which can have implications in developing strategies to effectively control PLS in peppers.

In vitro evaluation of the carcinogenic potential of perfluorinated chemicals.

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the major components of long-chain per- and polyfluorinated alkyl substances (PFAS), known for their chemical stability and environmental persistence. Even if PFOA and PFOS have been phased out or are limited in use, they still represent a concern for human and environmental health. Several studies have been per­formed to highlight the toxicological behavior of these chemicals and their mode of action (MoA). Data have suggested a causal association between PFOA or PFOS exposure and carcinogenicity in humans, but the outcomes of epidemiological studies showed some inconsistency. Moreover, the hypothesized MoA based on animal studies is considered not relevant for human cancer. To improve the knowledge on PFAS toxicology and contribute to the weight of evidence for the regu­latory classification of PFAS, we used the BALB/c 3T3 cell transformation assay (CTA), an in vitro model under consideration to be included in an integrated approach to testing and assessment for non-genotoxic carcinogens (NGTxCs). PFOS and PFOA were tested at several concentrations using a validated experimental protocol. Our results demonstrate that PFOA does not induce cell transformation, whereas PFOS exposure induced a concentration-related increase of type III foci. Malignant foci formation was triggered at PFOS concentrations equal to or higher than 50 ppm and was not directly associated with cytotoxicity or proliferation induction. The divergent CTA outcomes suggest that different molecular events could be responsible for the toxicological profiles of PFOS and PFOA, which were not fully captured in our study.

PFAS chemicals are known for their durability and resistance to heat, water, and oil. They are per­sistent in the environment and may pose health risks despite decreased use. This study explored PFOS and PFOA, two common PFAS chemicals, to understand their potential harm and cancer risk. To better understand how they might be harmful, we conducted a cell-based test that can resemble the carcinogenesis process in experimental animals. The test revealed PFOS, but not PFOA, can cause cancer-like changes, at levels of 50 parts per million or higher. This result suggests different PFAS chemicals affect cells differently, but we need more research to understand exactly how they work and how they might cause cancer. Understanding this could help regulate and reduce PFAS harmful effects. This research aligns with 3R principles by using cell-based tests as an alternative to animal testing, thereby promoting ethical research practices.

Migration Drives the Replacement of Xanthomonas perforans Races in the Absence of Widely Deployed Resistance.

Changes in Xanthomonas race and species composition causing bacterial spot of tomato have occurred throughout the world and are often associated with epidemics. Knowledge of bacterial population structure is key for resistance discovery and deployment. We surveyed Xanthomonas spp. composition from processing tomato fields in the Midwestern United States over a 4-year period between 2017 and 2020, compared these to strains collected previously, and found that X. perforans is currently the most prevalent species. We characterized 564 X. perforans isolates for sequence variation in avrXv3 to distinguish between race T3 and T4 and validated race designation using hypersensitive response (HR) assays for 106 isolates. Race T4 accounted for over 95% of X. perforans isolates collected in the Midwest between 2017 and 2020. Whole genome sequencing, Average Nucleotide Identity (ANI) analysis, core genome alignment and single nucleotide polymorphism (SNP) detection relative to a reference strain, and phylogenomic analysis suggest that the majority of Midwestern X. perforans strains collected between 2017 and 2020 were nearly identical, with greater than 99.99% ANI to X. perforans isolates collected from Collier County, Florida in 2012. These isolates shared a common SNP variant resulting an a premature stop codon in avrXv3. One sequenced isolate was identified with a deletion of avrXv3 and shared 99.99% ANI with a strain collected in Collier Co., Florida in 2006. A population shift to X. perforans T4 occurred in the absence of widely deployed resistance, with only 7% of tomato varieties tested having the resistant allele at the Xv3/Rx-4 locus. The persistence of nearly identical strains over multiple years suggests that migration led to the establishment of an endemic population. Our findings validate a genomics-based framework to track shifts in X. perforans populations due to migration, mutation, drift, or selection based on comparisons to 146 genomes.

The Secretive Liaison of Particulate Matter and SARS-CoV-2. A Hypothesis and Theory Investigation.

As the novel coronavirus disease sweeps across the world, there is growing speculation on the role that atmospheric factors may have played on the different distribution of SARS-CoV-2, and on the epidemiological characteristics of COVID-19. Knowing the role that environmental factors play in influenza virus outbreaks, environmental pollution and, in particular, atmospheric airborne (particulate matter, PM) has been considered as a potential key factor in the spread and mortality of COVID-19. A possible role of the PM as the virus carrier has also been debated. The role of PM in exacerbating respiratory and cardiovascular disease has been well recognized. Accumulating evidence support the hypothesis that PM can trigger inflammatory response at molecular, cellular and organ levels. On this basis, we developed the hypothesis that PM may play a role as a booster of COVID-19 rather than as a carrier of SARS-CoV-2. To support our hypothesis, we analyzed the molecular signatures detected in cells exposed to PM samples collected in one of the most affected areas by the COVID-19 outbreak, in Italy. T47D human breast adenocarcinoma cells were chosen to explore the global gene expression changes induced by the treatment with organic extracts of PM 2.5. The analysis of the KEGG's pathways showed modulation of several gene networks related to the leucocyte transendothelial migration, cytoskeleton and adhesion system. Three major biological process were identified, including coagulation, growth control and immune response. The analysis of the modulated genes gave evidence for the involvement of PM in the endothelial disease, coagulation disorders, diabetes and reproductive toxicity, supporting the hypothesis that PM, directly or through molecular interplay, affects the same molecular targets as so far known for SARS-COV-2, contributing to the cytokines storm and to the aggravation of the symptoms triggered by COVID-19. We provide evidence for a plausible cooperation of receptors and transmembrane proteins, targeted by PM and involved in COVID-19, together with new insights into the molecular interplay of chemicals and pathogens that could be of importance for sustaining public health policies and developing new therapeutic approaches.

Hazard assessment of air pollutants: The transforming ability of complex pollutant mixtures in the Bhas 42 cell model.

The use of in vitro alternative methods is a promising approach to characterize the hazardous properties of environmental chemical mixtures, including urban airborne particulate matter (PM). The aim of this study was to examine seasonal differences in the toxic and transforming potential of PM samples, by using the in vitro cell transformation assay in Bhas 42 cells for the prediction of potential carcinogenic effects. Bhas 42 cells are already initiated, and the v-Ha-ras transfection, together with genetic modification following the immortalization process, makes them a valuable model to study the late steps of cellular transformation leading to the acquisition of the malignant phenotype. Exposure to organic extracts of PM1 and PM2.5 induced dose-related effects. The transforming and cytotoxic properties are related to the amount of PM collected during the sampling campaign and associated with the concentrations of polycyclic aromatic hydrocarbons (PAHs) in the samples. All the samples induced cell transformation following prolonged exposure of 2 weeks. Our results support the utility of the in vitro top-down approach to characterise the toxicity of real mixtures, thereby supporting regulators in the decision-making process. The results also identify the need for appropriate assay selection within the in vitro testing strategy to address the complexity of the final adverse outcomes.

Co-carcinogenic effects of vitamin E in prostate.

A large number of basic researches and observational studies suggested the cancer preventive activity of vitamin E, but large-scale human intervention trials have yielded disappointing results and actually showed a higher incidence of prostate cancer although the mechanisms underlying the increased risk remain largely unknown. Here we show through in vitro and in vivo studies that vitamin E produces a marked inductive effect on carcinogen-bioactivating enzymes and a pro-oxidant status promoting both DNA damage and cell transformation frequency. First, we found that vitamin E in the human prostate epithelial RWPE-1 cell line has the remarkable ability to upregulate the expression of various phase-I activating cytochrome P450 (CYP) enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), giving rise to supraphysiological levels of reactive oxygen species. Furthermore, our rat model confirmed that vitamin E in the prostate has a powerful booster effect on CYP enzymes associated with the generation of oxidative stress, thereby favoring lipid-derived electrophile spread that covalently modifies proteins. We show that vitamin E not only causes DNA damage but also promotes cell transformation frequency induced by the PAH-prototype benzo[a]pyrene. Our findings might explain why dietary supplementation with vitamin E increases the prostate cancer risk among healthy men.

Molecular diversity and allergenic profiles of Alternaria spp. from desert environments in Arizona.

This study examined the genetic diversity of small-spored Alternaria species in the southwest desert of the USA by sampling 552 isolates from different habitats (soil and plant debris) in different locations (urban and an undisturbed desert). To estimate the genetic diversity, Amplified Fragment Length Polymorphism (AFLP) fingerprinting analysis was performed for all isolates. Strains representative of the sampled genotypic diversity (n = 125) were further characterized according their sporulation pattern and the capability to produce allergens. Morphological characterization assigned the majority of the strains to the Alternaria alternata and Alternaria tenuissima morpho-groups with only two isolates assigned to the Alternaria arborescens morpho-group. AFLP fingerprinting differentiated the A. arborescens morpho-groups, but could not distinguish between the A. alternata and A. tenuissima morpho-groups. Western blot analysis showed that a large number of allergenic proteins were produced by strains. These proteins were not specific for any morpho-group nor source of isolation. A hierarchical analysis of molecular variance was performed on the AFLP data to quantify molecular variation and partition this variation among sampled locations and habitat. No statistically significant differentiation among locations and habitat was detected indicating a lack of population structure across environments.

Identification of pathway-based toxicity in the BALB/c 3T3 cell model.

The particulate matter represents one of the most complex environmental mixtures, whose effects on human health and environment vary according to particles characteristics and source of emissions. The present study describes an integrated approach, including in vitro tests and toxicogenomics, to highlight the effects of air particulate matter on toxicological relevant endpoints. Air samples (PM2.5) were collected in summer and winter at different sites, representative of different levels of air pollution. Samples organic extracts were tested in the BALB/c 3T3 CTA at a dose range 1-12m(3). The effect of the exposure to the samples at a dose of 8m(3) on the whole-genome transcriptomic profile was also assessed. All the collected samples induced dose-related toxic effects in the exposed cells. The modulated gene pathways confirmed that toxicity was related to sampling season and sampling site. The analysis of the KEGG's pathways showed modulation of several gene networks related to oxidative stress and inflammation. Even if the samples did not induce cell transformation in the treated cells, gene pathways related to the onset of cancer were modulated as a consequence of the exposure. This integrated approach could provide valuable information for predicting toxic risks in humans exposed to air pollution.