Identification of microRNA-like RNAs in Cordyceps guangdongensis and their expression profile under differential developmental stages.

Cordyceps guangdongensis is a well-known fungus with high nutritional and medicinal value. The metabolite profile of C. guangdongensis is similar to that of Ophiocordyceps sinensis. In plants and animals, microRNAs play important roles in regulating gene expression at the post-transcriptional level. MicroRNA-like RNAs (milRNAs) have been documented in several macro-fungi. To comprehensively investigate the milRNAs in C. guangdongensis, three small RNA libraries from the differentially developmental stages were constructed. Twenty-six conserved milRNAs were identified, and 19 novel milRNA candidates were predicted. Among them, 20 milRNAs were differentially expressed across the developmental processes, and 12 milRNAs were verified using stem-loop quantitative real-time reverse transcription polymerase chain reaction. In addition, the potential target genes of milRNA were predicted to be involved in the development of fruiting bodies and metabolite biosynthesis. This study is the first to report the milRNAs of C. guangdongensis, and provides important insights into studies of milRNA regulation pathways in ascomycete fungi.

Mitogenome of Tolypocladium guangdongense.

Tolypocladium guangdongense is a high-value edible fungus with various medicinal and food safety properties. However, its evolutionary and genetic information is still limited. Mitochondrial genomes are potential models for molecular evolution and phylogenetic studies. In this study, we sequenced the complete mitogenome of T. guangdongense, demonstrating circular sequence of 46,102 bp, containing 14 standard protein-coding genes (PCGs), 2 ribosomal RNA subunit genes, and 28 tRNA genes. Phylogenetic analysis based on mitochondrial genes indicated that T. guangdongense was clustered into the Tolypocladium genus with high support value, based on the core PCG dataset. In addition, rps3 is also a suitable marker in the phylogenetic analysis in Hypocreales. Gene rearrangement analysis indicated that the gene order of PCGs was highly consistent in Hypocreales, and tRNA rearrangement events occurred in most species of Hypocreales; however, the rearrangement rates were not taxonomically correlated. Divergence time estimation based on the old fossil record and previous reports revealed that T. guangdongense originated approximately in the middle Cenozoic (42 Mya, 95% highest posterior density interval: 43-116) with the Tolypocladium genus differentiation. Our results provided more mitogenomic information of T. guangdongense and shed new insights into evolution of the Tolypocladium genus. KEY POINTS: • The general and unique features of T. guangdongense mitogenome are firstly reported. • Phylogenetic analysis further verified the taxonomic status of T. guangdongense. • Divergence time estimation provides more evolutionary information of T. guangdongense.

Fungal Systematics and Evolution: FUSE 5.

Thirteen new species are formally described: Cortinarius brunneocarpus from Pakistan, C. lilacinoarmillatus from India, Curvularia khuzestanica on Atriplex lentiformis from Iran, Gloeocantharellus neoechinosporus from China, Laboulbenia bernaliana on species of Apenes, Apristus, and Philophuga (Coleoptera, Carabidae) from Nicaragua and Panama, L. oioveliicola on Oiovelia machadoi (Hemiptera, Veliidae) from Brazil, L. termiticola on Macrotermes subhyalinus (Blattodea, Termitidae) from the DR Congo, Pluteus cutefractus from Slovenia, Rhizoglomus variabile from Peru, Russula phloginea from China, Stagonosporopsis flacciduvarum on Vitis vinifera from Italy, Strobilomyces huangshanensis from China, Uromyces klotzschianus on Rumex dentatus subsp. klotzschianus from Pakistan. The following new records are reported: Alternaria calendulae on Calendula officinalis from India; A. tenuissima on apple and quince fruits from Iran; Candelariella oleaginescens from Turkey; Didymella americana and D. calidophila on Vitis vinifera from Italy; Lasiodiplodia theobromae causing tip blight of Dianella tasmanica 'variegata' from India; Marasmiellus subpruinosus from Madeira, Portugal, new for Macaronesia and Africa; Mycena albidolilacea, M. tenuispinosa, and M. xantholeuca from Russia; Neonectria neomacrospora on Madhuca longifolia from India; Nothophoma quercina on Vitis vinifera from Italy; Plagiosphaera immersa on Urtica dioica from Austria; Rinodina sicula from Turkey; Sphaerosporium lignatile from Wisconsin, USA; and Verrucaria murina from Turkey. Multi-locus analysis of ITS, LSU, rpb1, tef1 sequences revealed that P. immersa, commonly classified within Gnomoniaceae (Diaporthales) or as Sordariomycetes incertae sedis, belongs to Magnaporthaceae (Magnaporthales). Analysis of a six-locus Ascomycota-wide dataset including SSU and LSU sequences of S. lignatile revealed that this species, currently in Ascomycota incertae sedis, belongs to Pyronemataceae (Pezizomycetes, Pezizales).

Two new species of Phylloporus from Bangladesh, with morphological and molecular evidence.

Two new species of Phylloporus from Bangladesh forests dominated by Shorea robusta are described and illustrated, based on morphological and molecular phylogenetic evidence. Phylloporus attenuatus is characterized by its wine-red to brownish-red pilei, decurrent lamellae that stain blue, downwards tapering stipe, a trichodermium pileipellis, and ellipsoidal-ovoid to ellipsoidal basidiospores (7-8 × 4-5 µm). Phylloporus catenulatus is characterized by its small-sized basidiomata with reddish-brown pilei, narrowly decurrent with distant lamellae that stain blue, a subepithelium pileipellis, and ellipsoidal to ellipsoidal-ovoid basidiospores (8-9.5 × 4-5 µm). Both new species of Phylloporus are distinctive from any known taxa based on phylogenetic analyses of partial sequences of the nuc rDNA internal transcribed spacer (ITS; including ITS1-ITS2 but not 5.8S) region, partial sequence of nuclear ribosomal large subunit (28S) domains D1 and D2, and translation elongation factor 1-α (TEF1α). Both species are compared with phenotypically similar taxa and illustrated with line drawings and photographs. A key to the species of Phylloporus known from Bangladesh is provided.

Genetic diversity analysis of Hypsizygus marmoreus with target region amplification polymorphism.

Hypsizygus marmoreus is an industrialized edible mushroom. In the present paper, the genetic diversity among 20 strains collected from different places of China was evaluated by target region amplification polymorphism (TRAP) analysis; the common fragment of TRAPs was sequenced and analyzed. Six fixed primers were designed based on the analysis of H. marmoreus sequences from GenBank database. The genomic DNA extracted from H. marmoreus was amplified with 28 TRAP primer combinations, which generated 287 bands. The average of amplified bands per primer was 10.27 (mean polymorphism is 69.73%). The polymorphism information content (PIC) value for TRAPs ranged from 0.32 to 0.50 (mean PIC value per TRAP primer combination is 0.48), which indicated a medium level of polymorphism among the strains. A total of 36 sequences were obtained from TRAP amplification. Half of these sequences could encode the known or unknown proteins. According to the phylogenetic analysis based on TRAP result, the 20 strains of H. marmoreus were classified into two main groups.

Anti-fatigue property of Cordyceps guangdongensis and the underlying mechanisms.

CONTEXT: Cordyceps guangdongensis T.H. Li, Q.Y. Lin & B. Song (Cordycipitaceae) is a nontoxic folk medicine and can be cultivated, with noticeable effects of anti-H9N2, life-prolonging and treating chronic renal failure. OBJECTIVE: The anti-fatigue effect of C. guangdongensis, possible mechanism and active constituent were investigated. MATERIALS AND METHODS: Treatment mice were treated with C. guangdongensis powder (0.455, 0.91 and 1.82 g/kg bw daily for low, middle and high doses, respectively); treatment rats were fed, respectively, with ethanol, petroleum ether, ethyl acetate, n-butanol, aqueous phase and hot water extract fractions, for 30 d. Forced swimming time to exhaustion, blood urea nitrogen (BUN) and hepatic glycogen (HG) levels of mice and blood lactic acid (BLC) levels of rats were determined. RESULTS: The swimming times to exhaustion of mice were very significantly (p < 0.01) longer in low-, middle- and high-dose groups (respectively 1.87-, 1.94- and 1.88-times), and significantly (p < 0.05) longer in the n-butanol fraction group (1.52-times), hot water extract group (1.88- times) and refined polysaccharide group (2.66-times) than in blank control; the BLC levels of rats were significantly (p < 0.05) lower in the ethyl alcohol partition group (84.8%), the n-butanol fraction group (84.0%) and the hot water extract group (84.4 %) than in blank. The BUN and HG levels were not significantly different. DISCUSSION AND CONCLUSION: Cordyceps guangdongensis can potently alleviate fatigue through reducing the accumulation of BLC; a functional constituent was the refined polysaccharide. This might become a new functional food for fatigue resistance.

[Study on biological characteristics of Cordyceps ramosa mycelia].

OBJECTIVE: To study the biological characteristics of Cordyceps ramosa mycelia. METHODS: The effects of temperature, pH, carbon source, nitrogen source and C/N ratio on the mycelial growth of Cordyceps ramosa were investigated. RESULTS: The optimum temperature, pH, carbon source, nitrogen source and C/N ratio for the mycelial growth were 25 degrees C, 5.0-7.0, soluble starch, yeast extract and 24:1, respectively. CONCLUSION: The results provide a reference for cultivation of Cordyceps ramosa.

Phylogeny and Taxonomic Revision of the Family Discinaceae (Pezizales, Ascomycota).

Species of Discinaceae are common macrofungi with a worldwide distribution. Some of them are commercially consumed, while a few others are reported as poisonous. Two genera were accepted in the family: the epigeous Gyromitra with discoid, cerebriform to saddle-shaped ascomata and the hypogeous Hydnotrya with globose or tuberous ascomata. However, due to discrepancies in their ecological behaviors, a comprehensive investigation of their relationship was not thoroughly explored. In this study, phylogenies of Discinaceae were reconstructed using sequence analyses of combined and separate three gene partitions (internal transcribed spacer [ITS], large subunit ribosomal DNA [LSU], and translation elongation factor [TEF]) with a matrix containing 116 samples. As a result, the taxonomy of the family was renewed. Eight genera were recognized: two of them (Gyromitra and Hydnotrya) were retained, three (Discina, Paradiscina, and Pseudorhizina) were revived, and three (Paragyromitra, Pseudodiscina, and Pseudoverpa) were newly established. Nine new combinations were made in four genera. Two new species in Paragyromitra and Pseudodiscina and an un-named taxon of Discina were described and illustrated in detail based on the materials collected from China. Furthermore, a key to the genera of the family was also provided. IMPORTANCE Taxonomy of the fungal family Discinaceae (Pezizales, Ascomycota) was significantly renewed on the basis of sequence analyses of internal transcribed spacer (ITS), large subunit ribosomal DNA (LSU), and translation elongation factor (TEF). Eight genera were accepted, including three new genera; two new species were described; and nine new combinations were made. A key to the accepted genera of the family is provided. The aim of this study is to deepen the understanding of the phylogenetic relationships among genera of the group, as well as the associated generic concepts.

New insights into the genus Gyroporus (Gyroporaceae, Boletales), with establishment of four new sections and description of five new species from China.

Species of Gyroporus from southern China were studied in this study. Based on morphology and molecular phylogenetic analyses of DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS), the nuclear ribosomal large subunit (nrLSU), and the mitochondrial adenosine triphosphate ATP synthase subunit 6 (atp6), Gyroporus was divided into four main branches in the phylogenetic tree, and four sections were firstly proposed i.e. Gyroporus sect. Castaneus, G. sect. Cyanescens, G. sect. Longicystidiatus and G. sect. Pallidus. Five new species, i.e. G. alboluteus, G. atrocyanescens, G. pseudolongicystidiatus, G. pallidus and G. subcaerulescens, were revealed from China, and their phylogenetic positions were also analysed. Among them, G. alboluteus and G. pallidus were nested into the sect. Pallidus, although morphologically similar to G. castaneus; G. atrocyanescens and G. subcaerulescens, with obvious cyanescent oxidation reactions, were nested into the sect. Cyanescens; and G. pseudolongicystidiatus characterised by its long cystidia and was nested into the sect. Longicystidiatus. The new species were formally described and illustrated in the present study, and a key to the sections and species of Gyroporus in China was provided.

Diversity of Cantharellus (Cantharellales, Basidiomycota) in China with Description of Some New Species and New Records.

Cantharellus is a well-known genus of edible mushrooms, belonging to the family Hydnaceae in the class Agaricomycetes. In this study, a phylogenetic overview of Cantharellus subg. Cinnabarinus and C. subg. Parvocantharellus in China is carried out with the description of four new species. Species description are based on morphological characters of basidiomata and phylogenetic analyses of multi-locus dataset of 28S + tef1 + rpb2. Among the new species, two species, C. chrysanthus and C. sinocinnabarinus, belong to C. subg. Cinnabarinus and two new species, C. convexus and C. neopersicinus, belong to C. subg. Parvocantharellus. Species delimitation characters of the new taxa are compared with closely related species. In addition, three new records of Cantharellus are reported for China: C. albovenosus and C. citrinus of subg. Cinnabarinus and C. koreanus of subg. Parvocantharellus. A key to the species of subg. Cinnabarinus in China was provided.

Emending Gymnopus sect. Gymnopus (Agaricales, Omphalotaceae) by including two new species from southern China.

Based on phylogenetic analyses, some newly studied Chinese mushroom specimens were found to represent two distinct species within the genus Gymnopus. Along with G.fusipes (sect. Gymnopus) they form a distinct clade with high support, although their macromorphological characters seem to be closer to members of Gymnopus sect. Levipedes or sect. Vestipedes (Collybiopsis). When examined in detail, their micromorphological characters, especially the type of pileipellis, support them as new members of G. sect. Gymnopus. Therefore, two new species, G.omphalinoides and G.schizophyllus, and the emended circumscription of sect. Gymnopus are proposed in this paper. Detailed morphological descriptions, colour photos, illustrations of the two new species, morphological comparisons with similar taxa and the molecular-phylogenetic analyses of the combined nrITS and nrLSU data are presented. A key to the known species of G. sect. Gymnopus is also presented.

Cordyceps guangdongensis lipid-lowering formula alleviates fat and lipid accumulation by modulating gut microbiota and short-chain fatty acids in high-fat diet mice.

Obesity has caused serious health and economic problems in the world. Cordyceps guangdongensis is a high-value macrofungus with broad application potential in the food and bio-medicine industry. This current study aimed to estimate the role of C. guangdongensis lipid-lowering compound formula (CGLC) in regulating fat and lipid accumulation, gut microbiota balance, short-chain fatty acid (SCFA) contents, and expression levels of genes involved in fat and lipid metabolism in high-fat diet (HFD) mice. The results showed that CGLC intervention markedly reduced body weights and fat accumulation in HFD mice, improved glucose tolerance and blood lipid levels, and decreased lipid droplet accumulation and fat vacuole levels in the liver. CGLC decreased the ratio of Firmicutes and Bacteroidetes and increased the relative abundances of Bacteroides (B. acidifaciens) and Bifidobacterium (B. pseudolongum). In addition, CGLC treatment significantly promoted the production of SCFAs and regulated the relative expression levels of genes involved in fat and lipid metabolism in liver. Association analysis showed that several species of Bacteroides and most of SCFAs were significantly associated with serum lipid indicators. These results suggested that CGLC is a novel candidate formulation for treating obesity and non-alcohol fatty liver by regulating gut microbiota, SCFAs, and genes involved in fat and lipid metabolism.

Performance of a Chest Radiograph AI Diagnostic Tool for COVID-19: A Prospective Observational Study.

Purpose: To conduct a prospective observational study across 12 U.S. hospitals to evaluate real-time performance of an interpretable artificial intelligence (AI) model to detect COVID-19 on chest radiographs. Materials and Methods: A total of 95 363 chest radiographs were included in model training, external validation, and real-time validation. The model was deployed as a clinical decision support system, and performance was prospectively evaluated. There were 5335 total real-time predictions and a COVID-19 prevalence of 4.8% (258 of 5335). Model performance was assessed with use of receiver operating characteristic analysis, precision-recall curves, and F1 score. Logistic regression was used to evaluate the association of race and sex with AI model diagnostic accuracy. To compare model accuracy with the performance of board-certified radiologists, a third dataset of 1638 images was read independently by two radiologists. Results: Participants positive for COVID-19 had higher COVID-19 diagnostic scores than participants negative for COVID-19 (median, 0.1 [IQR, 0.0-0.8] vs 0.0 [IQR, 0.0-0.1], respectively; P < .001). Real-time model performance was unchanged over 19 weeks of implementation (area under the receiver operating characteristic curve, 0.70; 95% CI: 0.66, 0.73). Model sensitivity was higher in men than women (P = .01), whereas model specificity was higher in women (P = .001). Sensitivity was higher for Asian (P = .002) and Black (P = .046) participants compared with White participants. The COVID-19 AI diagnostic system had worse accuracy (63.5% correct) compared with radiologist predictions (radiologist 1 = 67.8% correct, radiologist 2 = 68.6% correct; McNemar P < .001 for both). Conclusion: AI-based tools have not yet reached full diagnostic potential for COVID-19 and underperform compared with radiologist prediction.Keywords: Diagnosis, Classification, Application Domain, Infection, Lung Supplemental material is available for this article.. © RSNA, 2022.

Streptomyces caeruleatus sp. nov., with dark blue diffusible pigment.

An actinomycete, designated strain GIMN4.002(T), was isolated from a tomato rhizosphere soil sample in Guangzhou, China. The strain produces white aerial mycelium and dark blue diffusible pigment on Gause's synthetic agar, and microscopic observation revealed that it produces looped chains of spiny spores. Morphological and chemotaxonomic characteristics of the strain are typical of the genus Streptomyces. Melanin was produced and antibacterial activity was detected against Gram-positive micro-organisms, such as Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus. The 16S rRNA gene sequence of strain GIMN4.002(T) had highest similarity (99.4  %) to Streptomyces lincolnensis B91; however, DNA-DNA relatedness between strain GIMN4.002(T) and S. lincolnensis NBRC 13054(T) was only 32.17  %. Further, the morphological, physiological and biochemical characteristics of strain GIMN4.002(T) are distinct from S. lincolnensis and other species of the genus Streptomyces with which this strain has high 16S rRNA gene sequence similarity (98-99  %). On the basis of the physiological and molecular properties observed, it is proposed that strain GIMN4.002(T) represents a novel species of the genus Streptomyces, for which the name Streptomyces caeruleatus sp. nov. is proposed, with GIMN4.002(T) (=CCTCC M 208213(T) =NRRL B-24802(T)) as the type strain.

Peptide toxin components of Amanita exitialis basidiocarps.

Eight peptide toxins were isolated and purified from basidiocarps of Amanita exitialis with high performance liquid chromatography and were subjected to ultraviolet, nuclear magnetic resonance and mass spectrometry. We identified seven peptide toxins, α-amanitin, ß-amanitin, amaninamide, phallacin, phallacidin, phallisacin and desoxoviroidin. The molecular weight (729.5 Da) of the eighth compound did not match that of any reported Amanita toxins and, although the UV absorption spectrum indicated it to be a phallotoxin, further studies are required to identify this component. This is the first report of amaninamide, phallacin, phallisacin and desoxoviroidin in this lethal mushroom species.

Detection of Isocitrate Dehydrogenase Mutated Glioblastomas Through Anomaly Detection Analytics.

BACKGROUND: The rarity of Isocitrate Dehydrogenase mutated (mIDH) glioblastomas relative to wild-type IDH glioblastomas, as well as their distinct tumor physiology, effectively render them "outliers". Specialized tools are needed to identify these outliers. OBJECTIVE: To carefully craft and apply anomaly detection methods to identify mIDH glioblastoma based on radiomic features derived from magnetic resonance imaging. METHODS: T1-post gadolinium images for 188 patients and 138 patients were downloaded from The Cancer Imaging Archive's (TCIA) The Cancer Genome Atlas (TCGA) glioblastoma collection, and from the University of Minnesota Medical Center (UMMC), respectively. Anomaly detection methods were tested on glioblastoma image features for the precision of mIDH detection and compared to standard classification methods. RESULTS: Using anomaly detection training methods, we were able to detect IDH mutations from features in noncontrast-enhancing regions in glioblastoma with an average precision of 75.0%, 69.9%, and 69.8% using three different models. Anomaly detection methods consistently outperformed traditional two-class classification methods from 2 unique learning models (67.9%, 67.6%). The disparity in performances could not be overcome through newer, popular models such as neural networks (67.4%). CONCLUSION: We employed an anomaly detection strategy in the detection of IDH mutation in glioblastoma using preoperative T1 postcontrast imaging. We show these methods outperform traditional two-class classification in the setting of dataset imbalances inherent to IDH mutation prevalence in glioblastoma. We validate our results using an external dataset and highlight new possible avenues for radiogenomic rare event prediction in glioblastoma and beyond.

Multigene Phylogeny and Morphology Reveal Unexpectedly High Number of New Species of Cantharellus Subgenus Parvocantharellus (Hydnaceae, Cantharellales) in China.

The genus Cantharellus, commonly known as chanterelles, has recently been divided into six subgenera; however, wider sampling approaches are needed to clarify the relationships within and between these groups. A phylogenetic overview of Cantharellus subgenus Parvocantharellus in China was inferred based on the large subunit nuclear ribosomal RNA gene (nrLSU), the DNA-directed RNA polymerase II subunit 2 (rpb2), and the transcription elongation factor 1-alpha (tef1). A total of nine species from China were assigned to the subgenus, including seven novel species, namely Cantharellusaurantinus, C. austrosinensis, C. galbanus, C. luteolus, C. luteovirens, C. minioalbus, and C. sinominior, and two known species, namely C. albus and C. zangii. The detailed descriptions and illustrations were provided based on the newly obtained data, with the comparisons to closely related species. C. zangii was restudied based on the paratype specimens and multiple new collections from the type locality. Futhermore, the Indian species C. sikkimensis was identified as a synonym of C. zangii based on the morphological and molecular analyses. A key to the Chinese species belonging to the subg. Parvocantharellus is also provided.

Two new species of Phallus (Phallaceae) with a white indusium from China.

Two new Phallus species, P.cremeo-ochraceus and P.rigidiindusiatus were discovered in southwestern and southern China, respectively. Phalluscremeo-ochraceus is morphologically characterized by its cream to ochraceous receptacle, white to very slightly pinkish indusium, white to pinkish pseudostipe and white to slightly purplish pink volva. Phallusrigidiindusiatus is characterized by a white to yellowish white receptacle, a strongly rigid indusium usually without serrated margin and smaller basidiospores than those of P.serratus. Phylogenetic positions of the two species are located in two independent lineages respectively. Detailed descriptions, color photographs, illustrations and a key to the related species are presented.

Biosynthetic Pathway and the Potential Role of Melatonin at Different Abiotic Stressors and Developmental Stages in Tolypocladium guangdongense.

Melatonin, a bioactive compound and an important signaling molecule produced in plants and animals, is involved in many biological processes. However, its function and synthetic pathways in fungi are poorly understood. Here, the samples from Tolypocladium guangdongense, a highly valued edible fungus with functional food properties, were collected under different experimental conditions to quantify the levels of melatonin and its intermediates. The results showed that the intracellular melatonin content was markedly improved by Congo red (CR), cold, and heat stresses; the levels of intracellular melatonin and its intermediates increased at the primordial (P) and fruiting body (FB) stages. However, the levels of most intermediates exhibited a notable decrease under CR stress. Several genes related to melatonin synthesis, excluding AADC (aromatic-L-amino-acid decarboxylase), were markedly upregulated at an early stage of CR stress but downregulated later. Compared to the mycelial stage, those genes were significantly upregulated at the P and FB stages. Additionally, exogenous melatonin promoted resistance to several abiotic stressors and P formation in T. guangdongense. This study is the first to report melatonin biosynthesis pathway in macro-fungi. Our results should help in studying the diversity of melatonin function and melatonin-synthesis pathways and provide a new viewpoint for melatonin applications in the edible-medicinal fungus.

A Prospective Observational Study to Investigate Performance of a Chest X-ray Artificial Intelligence Diagnostic Support Tool Across 12 U.S. Hospitals.

Importance: An artificial intelligence (AI)-based model to predict COVID-19 likelihood from chest x-ray (CXR) findings can serve as an important adjunct to accelerate immediate clinical decision making and improve clinical decision making. Despite significant efforts, many limitations and biases exist in previously developed AI diagnostic models for COVID-19. Utilizing a large set of local and international CXR images, we developed an AI model with high performance on temporal and external validation. Objective: Investigate real-time performance of an AI-enabled COVID-19 diagnostic support system across a 12-hospital system. Design: Prospective observational study. Setting: Labeled frontal CXR images (samples of COVID-19 and non-COVID-19) from the M Health Fairview (Minnesota, USA), Valencian Region Medical ImageBank (Spain), MIMIC-CXR, Open-I 2013 Chest X-ray Collection, GitHub COVID-19 Image Data Collection (International), Indiana University (Indiana, USA), and Emory University (Georgia, USA). Participants: Internal (training, temporal, and real-time validation): 51,592 CXRs; Public: 27,424 CXRs; External (Indiana University): 10,002 CXRs; External (Emory University): 2002 CXRs. Main Outcome and Measure: Model performance assessed via receiver operating characteristic (ROC), Precision-Recall curves, and F1 score. Results: Patients that were COVID-19 positive had significantly higher COVID-19 Diagnostic Scores (median .1 [IQR: 0.0-0.8] vs median 0.0 [IQR: 0.0-0.1], p < 0.001) than patients that were COVID-19 negative. Pre-implementation the AI-model performed well on temporal validation (AUROC 0.8) and external validation (AUROC 0.76 at Indiana U, AUROC 0.72 at Emory U). The model was noted to have unrealistic performance (AUROC > 0.95) using publicly available databases. Real-time model performance was unchanged over 19 weeks of implementation (AUROC 0.70). On subgroup analysis, the model had improved discrimination for patients with "severe" as compared to "mild or moderate" disease, p < 0.001. Model performance was highest in Asians and lowest in whites and similar between males and females. Conclusions and Relevance: AI-based diagnostic tools may serve as an adjunct, but not replacement, for clinical decision support of COVID-19 diagnosis, which largely hinges on exposure history, signs, and symptoms. While AI-based tools have not yet reached full diagnostic potential in COVID-19, they may still offer valuable information to clinicians taken into consideration along with clinical signs and symptoms.