Analysis of the Mycotoxin Levels and Expression Pattern of SWN Genes at Different Time Points in the Fungus Slafractonia leguminicola.

The fungal plant pathogen Slafractonia leguminicola produces two mycotoxins that affect animals: slaframine, which causes slobbers, and swainsonine, which causes locoism. Slafractonia leguminicola contains the swainsonine-associated orthologous gene clusters, "SWN", which include a multifunctional swnK gene (NRPS-PKS hybrid), swnH1 and swnH2 (nonheme iron dioxygenase genes), swnN and swnR (reductase genes), and swnT (transmembrane transporter). In addition to these genes, two paralogs of swnK, swnK1 (paralog1) and swnk2 (paralog2), are found in S. leguminicola. cDNAs from total mRNA were isolated from the S. leguminicola mycelia grown in the culture plates as well as from leaves inoculated with the fungal mycelia at different time points, and expression pattern of the SWN genes were analyzed using RT-qPCR. The concentrations of swainsonine and slaframine production from this fungus at different time points were also examined using liquid chromatography-mass spectrometry. The timing of gene expression was similar in cultured fungus and inoculated leaves and agreed with our proposed biosynthetic pathway. Substantially more swainsonine was produced than slaframine during time course studies.

Interactions of beet leafhopper (Hemiptera: Cicadellidae), vector of beet curly top virus, and hemp in New Mexico.

The beet leafhopper, Circulifer tenellus (Baker 1896), is the sole vector of beet curly top virus (BCTV). Both the virus and the vector have very wide host ranges, including many crops and weeds. Industrial hemp (Cannabis sativa L.) has been reported as a host for both the virus and leafhopper in the past few years with the legal cultivation of the crop in the United States. This research assessed the interactions of the beet leafhopper and hemp in New Mexico by determining the natural infection of hemp with BCTV in 3 field plots in 2021 and 2022 and monitoring the numbers of leafhoppers using yellow sticky traps. The relative preference of beet leafhopper for hemp types and varieties of hemp was assessed using cafeteria-style choice tests. Higher numbers of beet leafhoppers were trapped in and around hemp fields in 2022 than in 2021 in all 3 locations. BCTV was found to infect all 3 types of hemp (cannabidiol or CBD, fiber, and grain) in 2022 in 1 location and only a single CBD variety of hemp in the other 2 locations. Two BCTV strains were identified in CBD hemp, while an additional BCTV strain was found infecting chile pepper grown at the same location.

MariClus: Your One-Stop Platform for Information on Marine Natural Products, Their Gene Clusters and Producing Organisms.

BACKGROUND: The marine environment hosts the vast majority of living species and marine microbes that produce natural products with great potential in providing lead compounds for drug development. With over 70% of Earth's surface covered in water and the high interaction rate associated with liquid environments, this has resulted in many marine natural product discoveries. Our improved understanding of the biosynthesis of these molecules, encoded by gene clusters, along with increased genomic information will aid us in uncovering even more novel compounds. RESULTS: We introduce MariClus (https://www.mariclus.com), an online user-friendly platform for mining and visualizing marine gene clusters. The first version contains information on clusters and the predicted molecules for over 500 marine-related prokaryotes. The user-friendly interface allows scientists to easily search by species, cluster type or molecule and visualize the information in table format or graphical representation. CONCLUSIONS: This new online portal simplifies the exploration and comparison of gene clusters in marine species for scientists and assists in characterizing the bioactive molecules they produce. MariClus integrates data from public sources, like GenBank, MIBiG and PubChem, with genome mining results from antiSMASH. This allows users to access and analyze various aspects of marine natural product biosynthesis and diversity.

Silencing of the Transmembrane Transporter (swnT) Gene of the Fungus Slafractonia leguminicola Results in a Reduction of Mycotoxin Transport.

Slafractonia leguminicola infects red clover and other legumes, causing black patch disease. This pathogenic fungus also produces two mycotoxins, slaframine and swainsonine, that are toxic to livestock grazing on clover hay or pasture infested with S. leguminicola. Swainsonine toxicosis causes locoism, while slaframine causes slobbers syndrome. The mechanism of toxin secretion by S. leguminicola is poorly understood. The aim of this research was to investigate the role of a putative transmembrane transporter, SwnT, in mycotoxin transport. The swnT gene was silenced by RNA interference using the silencing vector Psilent1, which included inverted repeat transgenes of swnT. This resulted in a significant reduction of swnT transcript levels compared with the controls. Silencing caused a decline in the active efflux of toxins from the mycelia to the media, as shown by LC-MS analysis. Transformants in which swnT was silenced showed higher concentrations of both toxins in the mycelia compared with the concentrations in the media. These transformants exhibited a visibly distinct phenotype with much thicker and shorter mycelia than in the wild type. These transformants were also unable to infect detached clover leaves, unlike the controls, suggesting that SwnT function may play an important role in pathogenesis in addition to mycotoxin transport. This research demonstrates the importance of this transporter to the secretion of mycotoxins for this phytopathogenic fungus.

Phylogenetic Comparison of Swainsonine Biosynthetic Gene Clusters among Fungi.

Swainsonine is a cytotoxic alkaloid produced by fungi. Genome sequence analyses revealed that these fungi share an orthologous gene cluster, SWN, necessary for swainsonine biosynthesis. To investigate the SWN cluster, the gene sequences and intergenic regions were assessed in organisms containing swnK, which is conserved across all fungi that produce swainsonine. The orders of fungi which contained orthologous swainsonine genes included Pleosporales, Onygenales, Hypocreales, Chaetothyriales, Xylariales, Capnodiales, Microthyriales, Caliciales, Patellariales, Eurotiales, and a species of the Leotiomycetes. SwnK and swnH2 genes were conserved across all fungi containing the SWN cluster; in contrast, swnT and swnA were found in a limited number of fungi containing the SWN cluster. The phylogenetic data suggest that in some orders that the SWN cluster was gained once from a common ancestor while in other orders it was likely gained several times from one or more common ancestors. The data also show that rearrangements and inversions of the SWN cluster happened within a genus as species diverged. Analysis of the intergenic regions revealed different combinations and inversions of open reading frames, as well as absence of genes. These results provide evidence of a complex evolutionary history of the SWN cluster in fungi.

Localization of the Swainsonine-Producing Chaetothyriales Symbiont in the Seed and Shoot Apical Meristem in Its Host Ipomoea carnea.

Several species of fungi from the orders Chaetothyriales and Pleosporales have been reported to produce swainsonine and be associated as symbionts with plants of the Convolvulaceae and Fabaceae, respectively. An endosymbiont belonging to the Chaetothyriales produces swainsonine and grows as an epibiont on the adaxial leaf surfaces of Ipomoea carnea, but how the symbiont passes through plant growth and development is unknown. Herein, different types of microscopy were used to localize the symbiont in seeds and in cross sections of plant parts. The symbiont was found in several tissues including the hilum, the sclereids, and the hypocotyl of seeds. In five-day old seedlings and mature plants, the symbiont was found in the shoot apical meristem (SAM) and the adaxial surface of immature folded leaves. The mycelia generally formed a close association with peltate glandular trichomes. This report provides further data explaining the relationship between the seed transmitted Chaetothyriales symbiont and Ipomoea carnea. These results provide a possible explanation for how this symbiont, and others like Periglandula may persist and are transmitted over time.

Genetic Relationships in the Toxin-Producing Fungal Endophyte, Alternaria oxytropis Using Polyketide Synthase and Non-Ribosomal Peptide Synthase Genes.

The legume Oxytropis sericea hosts a fungal endophyte, Alternaria oxytropis, which produces secondary metabolites (SM), including the toxin swainsonine. Polyketide synthase (PKS) and non-ribosomal peptide synthase (NRPS) enzymes are associated with biosynthesis of fungal SM. To better understand the origins of the SM, an unannotated genome of A. oxytropis was assessed for protein sequences similar to known PKS and NRPS enzymes of fungi. Contigs exhibiting identity with known genes were analyzed at nucleotide and protein levels using available databases. Software were used to identify PKS and NRPS domains and predict identity and function. Confirmation of sequence for selected gene sequences was accomplished using PCR. Thirteen PKS, 5 NRPS, and 4 PKS-NRPS hybrids were identified and characterized with functions including swainsonine and melanin biosynthesis. Phylogenetic relationships among closest amino acid matches with Alternaria spp. were identified for seven highly conserved PKS and NRPS, including melanin synthesis. Three PKS and NRPS were most closely related to other fungi within the Pleosporaceae family, while five PKS and PKS-NRPS were closely related to fungi in the Pleosporales order. However, seven PKS and PKS-NRPS showed no identity with fungi in the Pleosporales or the class Dothideomycetes, suggesting a different evolutionary origin for those genes.

Molecular Characterization of a Fungal Ketide Synthase Gene Among Swainsonine-Producing Alternaria Species in the USA.

Locoweeds are toxic leguminous plants in Astragalus and Oxytropis genera that contain fungal endophytes of Alternaria section Undifilum species. These fungi produce swainsonine, an alkaloid α-mannosidase inhibitor that causes a neurological syndrome, locoism in grazing animals. A SWN gene cluster has been identified in many swainsonine-producing fungi. The swnK gene, which is an essential component of the swainsonine biosynthetic pathway, encodes a polyketide synthase-nonribosomal peptide synthase (PKS-NRPS). To determine if swnK was conserved among Alternaria section Undifilum endophytes of locoweed, the sequence of the KS region of swnK was compared between various swainsonine-producing fungi. The internal transcribed spacer (ITS), and glyceraldehyde-3-phosphate dehydrogenase (GPD) regions from the same fungi were also assessed. Sequences were examined at the nucleotide and protein levels. Alternaria oxytropis, A. fulva, A. cinerea, and Alternaria sp. from Swainsona species produced distinct clades for all multigene data sets. swnK-KS sequence did not differ among fungi isolated from Astragalus mollissimus varieties or A. lentiginosus varieties. The swnK-KS amino acid sequence was essentially identical among all swainsonine-producing Alternaria sp. Two low swainsonine-producing fungi, Alternaria bornmuelleri and A. gansuense, clustered together, as did non-pathogen Alternaria endophytes. The swnK-KS sequence comparisons were effective in identifying swainsonine production capability and differentiating among swainsonine-producing fungal species.

Physio-biochemical and ultrastructural impact of (Fe3O4) nanoparticles on tobacco.

BACKGROUND: Because of their broad applications in our life, nanoparticles are expected to be present in the environment raising many concerns about their possible adverse effects on the ecosystem of plants. The aim of this study was to examine the effect of different sizes and concentrations of iron oxide nanoparticles [(Fe3O4) NPs] on morphological, physiological, biochemical, and ultrastructural parameters in tobacco (Nicotiana tabacum var.2 Turkish). RESULTS: Lengths of shoots and roots of 5 nm-treated plants were significantly decreased in all nanoparticle-treated plants compared to control plants or plants treated with any concentration of 10 or 20 nm nanoparticles. The photosynthetic rate and leaf area were drastically reduced in 5 nm (Fe3O4) NP-treated plants of all concentrations compared to control plants and plants treated with 10 or 20 nm (Fe3O4) NPs. Accumulation of sugars in leaves showed no significant differences between the control plants and plants treated with iron oxide of all sizes and concentrations. In contrast, protein accumulation in plants treated with 5 nm iron oxide dramatically increased compared to control plants. Moreover, light and transmission electron micrographs of roots and leaves revealed that roots and chloroplasts of 5 nm (Fe3O4) NPs-treated plants of all concentrations were drastically affected. CONCLUSIONS: The size and concentration of nanoparticles are key factors affecting plant growth and development. The results of this study demonstrated that the toxicity of (Fe3O4) NPs was clearly influenced by size and concentration. Further investigations are needed to elucidate more about NP toxicity in plants, especially at the molecular level.

Identification, Characterization, Pathogenicity, and Distribution of Verticillium alfalfae in Alfalfa Plants in China.

Verticillium wilt caused by Verticillium alfalfae results in severe production losses in alfalfa crops and is a Class A quarantined disease in China. During 2015 to 2017, 365 alfalfa fields from 21 locations in six provinces were surveyed, and 45 fields from three closely located sites in Gansu, China were found to have alfalfa plants with symptoms typical of Verticillium wilt, with disease incidence of 12.6 to 53.6%. Isolates were identified to species using morphological characteristics and a maximum likelihood phylogeny of the concatenated partial sequences of actin, elongation factor, glyceraldehyde-3-phosphate dehydrogenase, and tryptophan synthase gene regions of Verticillium isolates. Isolation incidence was 93.9% from roots, 71.7% from stems, 66.1% from petioles, and 32.2% from leaves of field-infected plants, indicative of systemic disease and sporadic distribution of this pathogen. In greenhouse tests, the pathogen infected seedlings and colonized vascular tissues when inoculated on seeds, on root tips, in soil, or in injured, but not uninjured, aerial tissues, causing systemic symptoms like those in the field and significant losses. Pathogenicity testing also revealed that five locally grown perennial legumes (stylo, milkvetch, sainfoin, white clover, and red clover) could host V. alfalfae, with a high virulence to milkvetch, sainfoin, and stylo. This study confirmed that V. alfalfae has become established in some regions of Gansu, China and that is a risk to the alfalfa industry in China.

Time-course metabolic profiling in alfalfa leaves under Phoma medicaginis infection.

Information on disease process and pathogenicity mechanisms is important for understanding plant disease. Spring black stem and leaf spot caused by the necrotrophic pathogen Phoma medicaginis var. medicaginis Malbr. & Roum causes large losses to alfalfa. However, till now, little is known about alfalfa-P. medicagnis interactions and the pathogenicity mechanisms of the pathogen. Here, alfalfa inoculated with P. medicaginis was subjected to GC-MS based metabolic profiling. The metabolic response in P. medicaginis-inoculated and mock-inoculated alfalfa leaves was assessed at 2, 4, 6, 8, 12, 16, 20, 24, 26 and 28 days post inoculation. In total, 101 peaks were detected in the control and inoculated groups, from which 70 metabolites were tentatively identified. Using multivariate analysis, 16 significantly regulated compounds, including amino acids, nitrogen-containing compounds and organic acids, polyols, fatty acids, and sugars were tentatively identified (Variable importance values, VIP>1.0 and p <0.05). Among these metabolites, the levels of malate, 5-oxoproline, palmitic acid and stearic acid were increased significantly in P. medicaginis-infected alfalfa leaves compared to the controls. In contrast, the levels ofγ-aminobutyric acid and 2-pyrrolidinone were significantly decreased in infected leaves compared to the controls. Further metabolic pathway analysis of the 16 significantly regulated compounds demonstrated that glycolysis, the tricarboxylic acid cycle, and ß-oxidation of fatty acids were significantly induced in the alfalfa leaves at later stages of P. medicaginis infection. The strong induction of tricarboxylic acid cycle pathways at later infection stages caused by the pathogen may induce senescence in the alfalfa leaves, leading to plant death. However, intermediate metabolites of these metabolic pathways, and inositol phosphate, glutathione, the metabolic pathways of some amino acids accumulated rapidly and strongly at early stages of infection, which may enhance the ability of alfalfa to resist necrotrophic P. medicaginis disease. Understanding metabolic pathways is essential for understanding pathogenesis.

RNAi-mediated down-regulation of a melanin polyketide synthase (pks1) gene in the fungus Slafractonia leguminicola.

The fungus Slafractonia leguminicola, the causal agent of blackpatch disease of legumes produces two mycotoxins slaframine and swainsonine, causing slobbers' symptoms and locoism of grazing animals, respectively. The genetics of this important fungus is poorly understood. This work aimed to develop a genetic transformation system and evaluate the efficacy of RNA interference (RNAi) in S. leguminicola. In this study, S. leguminicola was transformed using a PEG-mediated method with a fungal construct that carries a hygromycin resistance cassette. To assess the use of RNAi, a silencing construct pSilentPKS1-AS was constructed which includes inverted repeat transgenes of the polyketide synthase gene (pks1) that is involved in melanin biosynthesis. Transformation of S. leguminicola with the IRT pks1 vector decreased pks1 transcripts levels 82-92% in knockdown mutants when compared with the wild type and was accompanied with a reduction in melanin and swainsonine production. These results demonstrate that RNAi can be a useful tool for studying gene function in S. leguminicola.

Swainsonine Biosynthesis Genes in Diverse Symbiotic and Pathogenic Fungi.

Swainsonine-a cytotoxic fungal alkaloid and a potential cancer therapy drug-is produced by the insect pathogen and plant symbiont Metarhizium robertsii, the clover pathogen Slafractonia leguminicola, locoweed symbionts belonging to Alternaria sect. Undifilum, and a recently discovered morning glory symbiont belonging to order Chaetothyriales. Genome sequence analyses revealed that these fungi share orthologous gene clusters, designated "SWN," which included a multifunctional swnK gene comprising predicted adenylylation and acyltransferase domains with their associated thiolation domains, a ß-ketoacyl synthase domain, and two reductase domains. The role of swnK was demonstrated by inactivating it in M. robertsii through homologous gene replacement to give a ∆swnK mutant that produced no detectable swainsonine, then complementing the mutant with the wild-type gene to restore swainsonine biosynthesis. Other SWN cluster genes were predicted to encode two putative hydroxylases and two reductases, as expected to complete biosynthesis of swainsonine from the predicted SwnK product. SWN gene clusters were identified in six out of seven sequenced genomes of Metarhzium species, and in all 15 sequenced genomes of Arthrodermataceae, a family of fungi that cause athlete's foot and ringworm diseases in humans and other mammals. Representative isolates of all of these species were cultured, and all Metarhizium spp. with SWN clusters, as well as all but one of the Arthrodermataceae, produced swainsonine. These results suggest a new biosynthetic hypothesis for this alkaloid, extending the known taxonomic breadth of swainsonine producers to at least four orders of Ascomycota, and suggest that swainsonine has roles in mutualistic symbioses and diseases of plants and animals.

A Re-examination of the Taxonomic Status of Embellisia astragali.

The plant pathogen Embellisia astragali causes a disease termed yellow stunt and root rot in standing milk-vetch (Astragalus adsurgens). The genus Embellisia and 12 allied genera were redefined as being synonymous with Alternaria in 2013. However, that taxonomic revision did not include E. astragali. The objective of this study was to determine the taxonomic status of this fungal species by comparing its morphological and molecular characteristics with those of other species. A phylogenetic tree developed based on the sequences of 3 loci (GPD, RPB2, and TEF-1) strongly supported the placement of E. astragali in Alternaria sect. Undifilum. The fungus also exhibited germ tubes and mycelia with a 'wavy' appearance on potato carrot agar, which is a characteristic of sect. Undifilum. Embellisia astragali was described here as Alternaria gansuense comb. nov. because the species epithet "astragali" was already occupied.

A search for the phylogenetic relationship of the ascomycete Rhizoctonia leguminicola using genetic analysis.

Rhizoctonia leguminicola, which causes fungal blackpatch disease of legumes and other plants, produces slaframine and swainsonine that are largely responsible for causing salivation, lacrimation, frequent urination, and diarrhea in grazing animals including cattle, sheep, and horses. The original identification of R. leguminicola was based only on morphological characters of the fungal mycelia in cultures because of the lack of fungal genetic markers. Recent investigations suggested that R. leguminicola does not belong to genus Rhizoctonia and is instead a member of the ascomycetes, necessitating an accurate reclassification. The objective of this study was to use both genetic and morphological characters of R. leguminicola to find taxonomic placement of this pathogen within ascomycetes. Internal transcribed spacer region (ITS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) encoding gene were amplified from R. leguminicola isolates by PCR using universal primers and sequencing. Rhizoctonia leguminicola ITS and gpd sequences were aligned with other fungal sequences of close relatives, and phylogenetic trees were constructed using neighbor-joining and parsimony analyses. Rhizoctonia leguminicola isolates were clustered within a clade that contains several genera of ascomycetes belonging to the class dothideomycetes. We suggest that the fungus is misidentified in the genus Rhizoctonia and propose its reclassification in a new genus within the phylum Ascomycota.

Production of the alkaloid swainsonine by a fungal endophyte in the host Swainsona canescens.

Legumes belonging to the Astragalus, Oxytropis, and Swainsona genera have been noted by ranchers in the Americas, Asia, and Australia to cause a neurologic disease often referred to as locoism or peastruck. The toxin in these legumes is swainsonine, an α-mannosidase and mannosidase II inhibitor. Recent research has shown that in Astragalus and Oxytropis species swainsonine is produced by a fungal endophyte belonging to the Undifilum genus. Here Swainsona canescens is shown to harbor an endophyte that is closely related to Undifilum species previously cultured from locoweeds of North America and Asia. The endophyte produces swainsonine in vitro and was detected by PCR and culturing in S. canescens. The endophyte isolated from S. canescens was characterized as an Undifilum species using morphological and phylogenetic analyses.

Potential role for saccharopine reductase in swainsonine metabolism in endophytic fungus, Undifilum oxytropis.

Locoweed plants in the southwestern United States often harbour a slow-growing endophytic fungus, Undifilum oxytropis (Phylum: Ascomycota; Order: Pleosporales), which produces a toxic alkaloid, swainsonine. Consumption of U. oxytropis by grazing animals induces a neurological disorder called locoism for which the toxic alkaloid swainsonine has been reported to be the causal agent. Little is known about the biosynthetic pathway of swainsonine in endophytic fungi, but previous studies on non-endophytic ascomycetous fungi indicate that pipecolic acid and saccharopine are key intermediates. We have used degenerate primers, Rapid amplification of cDNA ends (RACE)-PCR and inverse PCR to identify the gene sequence of U. oxytropis saccharopine reductase. To investigate the role of this gene product in swainsonine metabolism, we have developed a gene deletion system for this slow-growing endophyte based on our recently established transformation protocol. A strain of U. oxytropis lacking saccharopine reductase had decreased levels of saccharopine and lysine along with increased accumulation of pipecolic acid and swainsonine. Thus, saccharopine reductase influences the accumulation of swainsonine and its precursor, pipecolic acid, in U. oxytropis.

Performance of creatinine and cystatin C GFR estimating equations in an HIV-positive population on antiretrovirals.

OBJECTIVE: To evaluate the performance of Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) creatinine, cystatin C, and creatinine-cystatin C estimating equations in HIV-positive patients. METHODS: We evaluated the performance of the Modification of Diet in Renal Disease (MDRD) Study and CKD-EPI creatinine 2009, CKD-EPI cystatin C 2012, and CKD-EPI creatinine-cystatin C 2012 glomerular filtration rate (GFR) estimating equations compared with GFR measured using plasma clearance of iohexol in 200 HIV-positive patients on stable antiretroviral therapy. Creatinine and cystatin C assays were standardized to certified reference materials. RESULTS: Of the 200 participants, median (IQR) CD4 count was 536 (421) and 61% had an undetectable HIV viral load. Mean (SD) measured GFR (mGFR) was 87 (26) mL/min per 1.73 m. All CKD-EPI equations performed better than the MDRD Study equation. All 3 CKD-EPI equations had similar bias and precision. The cystatin C equation was not more accurate than the creatinine equation. The creatinine-cystatin C equation was significantly more accurate than the cystatin C equation, and there was a trend toward greater accuracy than the creatinine equation. Accuracy was equal or better in most subgroups with the combined equation compared to either alone. CONCLUSIONS: The CKD-EPI cystatin C equation does not seem to be more accurate than the CKD-EPI creatinine equation in patients who are HIV-positive, supporting the use of the CKD-EPI creatinine equation for routine clinical care for use in North American populations with HIV. The use of both filtration markers together as a confirmatory test for decreased estimated GFR based on creatinine in individuals who are HIV-positive requires further study.

Seasonal changes in Undifilum colonization and swainsonine content of locoweeds.

Locoweeds (Astragalus and Oxytropis) are leguminous plants that are toxic due to a symbiotic association with the endophytic fungus Undifilum oxytropis. The fungus produces the alkaloid swainsonine, an α-mannosidase-inhibitor that causes serious damage to mammals when consumed. A real-time PCR technique was developed to quantify the colonization extent of Undifilum in locoweeds and to compare it to the swainsonine concentration in the plants. Amplification of the endophyte nuclear ITS region allowed reliable quantification of Undifilum DNA from field plants and in vitro cultures. Swainsonine concentration was highly correlated (ρ = 0.972, P < 0.001) with the proportion of Undifilum DNA during the first 4 weeks of in vitro culture growth. Species of Astragalus and Oxytropis were sampled seasonally in New Mexico and Colorado for two years. High swainsonine concentration in plant samples was associated with high levels of endophyte DNA, except in plant reproductive tissues.