The locoweed endophyte Alternaria oxytropis affects root development in Arabidopsis in vitro through auxin signaling and polar transport.

Locoweeds are leguminous forbs known for their toxicity to livestock caused by the endophytic fungi Alternaria sect. Undifilum. Unlike the defensive mutualisms reported in many toxin-producing endophytes and their plant hosts, the benefits that A. sect. Undifilum can confer to it host plants remains unclear. Here, we conducted physiological and genetic analyses to show that A. (sect. Undifilum) oxytropis influences growth, especially root development, in its locoweed host Oxytropis ochrocephala and Arabidopsis. The presence of A. oxytropis significantly decreased primary root length while increasing the numbers of lateral roots and root hairs, and increasing plant leaf area and fresh weight. The fungus also increased the concentrations of plant endogenous auxin, and the expression of key genes for auxin biosynthesis, signaling, and transport. These effects on root development were abolished in mutants deficient in auxin signaling and polar transport. Alternaria oxytropis down-regulated expression of PIN1 but increased expression of PIN2, PIN7, and AUX1, which might reflect alterations in the spatial accumulation of auxin responsible for the changes in root architecture. Plant growth was insensitive to A. oxytropis when naphthylphthalamic acid was applied. Our findings indicate a function of A. oxytropis in promoting the growth and development of Arabidopsis via the regulation of auxin, which in turn suggests a possible role in benefiting its locoweed hosts via a process independent of its toxin production.

Alternaria gansuense, a Plant Systematic Fungal Pathogen Producing Swainsonine in Vivo and in Vitro.

Astragalus adsurgens (A. adsurgens), which is considered a forage in China, grows widely in Eurasia and North America. However, Alternaria gansuense (A. gansuense) (synonym: Embellisia astragali) systematically infects A. adsurgens, producing swainsonine (SW), which poisons domesticated animals. In this study, we hypothesized that the A. gansuense SW-producing fungus is morphologically and molecularly related to the locoweed endophyte, Alternaria oxytropis (A. oxytropis), which systematically grows in host plants. Therefore, pure cultures of the fungi from diseased plants or endophytic interactions were collected from fields and assayed for SW via high-performance liquid chromatography linked to mass spectroscopy (HPLC-MS). The production of SW was also detected in A. adsurgens, A. oxytropis and diseased plants by assaying for the presence of the ß-ketoacyl synthase (KS) gene, which is required for SW synthesis. Diseased A. adsurgens and pure cultures of A. gansuense have SW and the healthy-looking A. adsurgens plants also contained SW, probably because they were infected with A. gansuense. Therefore, A. adsurgens-infected A. gansuense are not safe for livestock consumption.

Systematical NMR analysis of swainsonine, a mycotoxin from endophytic fungus Alternaria oxytropis.

Swainsonine (SW, 1), a unique indolizine with poly-hydroxyl groups, was re-isolated from the plant endophytic fungus Alternaria oxytropis. The structure (including planar structure and relative configuration) was systematically elucidated by NMR spectra (including 1 H, 13 C, 1 H-1 H COSY, HMQC, HMBC, and NOESY spectra in DMSO-d6 and in CD3 OD); 1 H NMR spectra of the modified Mosher's products were first used to determine the absolute configuration of SW. More importantly, the complex coupled features of H-7α, H-7ß, and H-6α in the 1 H NMR spectrum of (1) were analyzed in details, which will provide aids for the planar and relative configuration determination of analogs.

Host Genotype and Precipitation Influence of Fungal Endophyte Symbiosis and Mycotoxin Abundance in a Locoweed.

Many plant endophytes produce mycotoxins, but how host genetic variation influences endophyte colonization and mycotoxin production under natural conditions is poorly understood. This interaction has not been fully considered in many previous studies which used controlled experiments with agronomic or model plant species. Here, we investigated this interaction in a naturally occurring forb (a locoweed species) Oxytropis ochrocephala, its symbiotic endophyte Alternaria oxytropis, and the mycotoxin swainsonine. Host genetic variation was characterized by microsatellite markers. Endophyte infection rate and swainsonine levels were determined by PCR and HPLC, respectively. Genetic markers defined two distinct host populations and revealed that host genetics were significantly correlated with geographical location, elevation, and precipitation. As the host diverged, symbiotic interactions were reduced or failed to produce detectable swainsonine in one host population. Host genotype and precipitation had a significant impact in shaping swainsonine production at the population level. This study highlights the effect of host genotype in influencing this interaction in locoweeds.

Application of Toxigenic Alternaria oxytropis to Soybeans and its Effect on Swainsonine Detection in Different Environments.

Alternaria oxytropis is an endophytic fungus of locoweeds that synthesizes swainsonine toxin. In this work, we evaluated the effect of A. oxytropis on soybean seedlings and quantified swainsonine in different culture conditions. Soybean (Glycine max) seeds were co-cultured with A. oxytropis (at different concentrations of mycelial suspensions) in agar media and soil culture, and swainsonine was assayed using LC-MS/MS. The results showed evidence that A. oxytropis infected soybean seedlings produced detectable swainsonine in agar culture while the toxin was undetectable or below the detection limit (0.006% of swainsonine dry weight) in soil media even at higher concentrations of the fungus. These results suggest that swainsonine detection is highly dependent on culture conditions and that soybeans co-cultured with A. oxytropis in soil could potentially be used to limit toxin production.

A Screen for Swainsonine in Select North American Astragalus Species.

Swainsonine is found in several plant species worldwide, and causes severe toxicosis in livestock grazing these plants, leading to a chronic condition characterized by weight loss, altered behavior, depression, decreased libido, infertility, and death. Swainsonine has been detected in 13 North American Astragalus species of which eight belong to taxa in four taxonomic sections, the Densifolii, Diphysi, Inflati, and Trichopodi. These sections belong to two larger groups representing several morphologically related species, the Pacific Piptolobi and the small-flowered Piptolobi. The objective of this study was to screen the other 31 species for swainsonine in sections Densifolii, Diphysi, Inflati, and Trichopodi previously not known to contain swainsonine. Furthermore, to broaden the scope further, 21 species within the 8 sections of the Pacific Piptolobi and the small flowered Piptolobi were screened for swainsonine. Swainsonine was detected for the first time in 36 Astragalus taxa representing 29 species using liquid and gas chromatography coupled with mass spectrometry. Several taxonomic sections were highly enriched in species that contain swainsonine while others were not. A systematic examination for swainsonine in these species will provide important information on the toxic risk of these species and may be a valuable reference for diagnosticians and land managers.

Identification and distribution of a fungal endosymbiotic Alternaria species (Alternaria section Undifilum sp.) in Astragalus garbancillo tissues.

Plants belonging to the genera Astragalus, Oxytropis, Ipomoea, Sida, and Swainsona often contain the toxin swainsonine (SW) produced by an associated fungal symbiont. Consumption of SW-containing plants causes a serious neurological disorder in livestock, which can be fatal. In this study, a fungal endophyte, Alternaria section Undifilum, was identified in Astragalus garbancillo seeds, using polymerase chain reaction (PCR) followed by direct sequencing. In seeds, the SW concentrations were about 4 times higher than in other parts of the plant. Furthermore, microscopic examination demonstrated that the fungus mycelium grows inside the petioles and stems, on the outer surface and inside the mesocarp of the fruit, in the mesotesta and endotesta layers of the seed coat, and inside the endosperm of the seeds. Our results support the notion that the SW-producing fungus is vertically transmitted in the host plant A. garbancillo.

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.

Assembly of high-quality genomes of the locoweed Oxytropis ochrocephala and its endophyte Alternaria oxytropis provides new evidence for their symbiotic relationship and swainsonine biosynthesis.

Locoweeds are perennial forbs poisonous to livestock and cause extreme losses to animal husbandry. Locoweed toxicity is attributed to the symbiotic endophytes in Alternaria sect. Undifilum, which produce a mycotoxin swainsonine (SW). We performed a de novo whole genome sequencing of the most common locoweed in China, Oxytropis ochrocephala (2n = 16), and assembled a high-quality, chromosome-level reference genome. Its genome size is 958.83 Mb with 930.94 Mb (97.09%) anchored and oriented onto eight chromosomes, and 31,700 protein-coding genes were annotated. Phylogenetic and collinearity analysis showed it is closely related to Medicago truncatula with a pair of large interchromosomal rearrangements, and both species underwent a whole-genome duplication event. We also derived the genome of A. oxytropis at 74.48 Mb with a contig N50 of 8.87 Mb and 10,657 protein-coding genes, and refined the genes of SW biosynthesis. Multiple Alternaria species containing the swnK gene were grouped into a single clade, but in other genera, swnK's homologues are diverse. Resequencing of 41 A. oxytropis strains revealed one SNP in the SWN cluster causing changes in SW concentration. Comparing the transcriptomes of symbiotic and nonsymbiotic interactions identified differentially expressed genes (DEGs) linked to defence and secondary metabolism in the host. Within the endophyte DEGs were linked to cell wall degradation, fatty acids and nitrogen metabolism. Symbiosis induced the upregulation of most of the SW biosynthetic genes. These two genomes and relevant sequencing data should provide valuable genetic resources for the study of the evolution, interaction, and SW biosynthesis in the symbiont.

Intoxication of llamas by Astragalus punae in Argentina.

Several plants that contain indolizidine alkaloids, including swainsonine, are toxic to livestock, causing dysfunctional lysosomes and storage disease. Swainsonine induces a neurovisceral disease, known as locoism, in sheep, goats, and cattle, which occurs in several parts of the world, including, but not limited to, the western United States, China, and parts of Australia. In South America, locoism has been described in the Andean region of Argentina affecting sheep, cattle, and llamas. Intoxication by consumption of Astragalus punae was suspected in 4 llamas in Jujuy Province, northwestern Argentina. The grazing area contained abundant specimens of A. punae. The clinical course was ~15 d, and included moderate ataxia, incoordination of hindlimbs, and progressive loss of body condition. Microscopically, fine cytoplasmic microvacuolation was observed in the proximal convoluted renal tubules. Ultrastructurally, these changes consisted of severely dilated lysosomes. Swainsonine was detected in stem and leaf samples of A. punae at a concentration of 0.06%. Based on clinical history and signs, histologic and ultrastructural changes, and plant analysis, a diagnosis of swainsonine toxicosis caused by consumption of A. punae was made, which has not been reported previously, to our knowledge.

A comparison of alternative sample preparation procedures for the analysis of swainsonine using LC-MS/MS.

INTRODUCTION: Swainsonine, a polyhydroxy indolizidine alkaloid and known glycosidase inhibitor, is found in a number of different plants that cause a lysosomal storage disease known as locoism in the western USA. Most recently swainsonine has been analysed by LC-MS/MS after sample extraction and preparation from ion-exchange resins. OBJECTIVE: To compare previously published sample preparation procedures with several new alternative procedures to provide methods using either commercially available solid-phase extraction equipment or procedures which significantly reduce sample preparation time. METHODOLOGY: A previously reported and validated sample preparation method using ion-exchange resin was compared with methods using a commercially available solid-phase extraction cartridge, a solvent partitioning procedure or a single solvent extraction procedure using one of two solvents. Twenty different plant samples of varying swainsonine concentrations were prepared in triplicate and analysed by LC-MS/MS. The measured concentration of swainsonine was then statistically compared between methods. RESULTS: There were no statistically significant differences found between four of the five different sample preparation methods tested. CONCLUSION: A commercially available SPE cartridge can be used to replace the previously used ion-exchange resin for swainsonine analysis. For very rapid analyses the SPE procedure can be eliminated and a simple, single solvent extraction step used for sample preparation.

Swainsoninine concentrations and endophyte amounts of Undifilum oxytropis in different plant parts of Oxytropis sericea.

Locoweeds are Astragalus and Oxytropis species that contain the toxic alkaloid swainsonine. Swainsonine accumulates in all parts of the plant with the highest concentrations found in the above ground parts. A fungal endophyte, Undifilum oxytropis, found in locoweed plant species, is responsible for the synthesis of swainsonine. By using quantitative PCR, the endophyte can be quantified in locoweed species. Endophyte amounts differ between plant parts and in some instances do not mirror the concentrations of swainsonine in the corresponding parts. Two groups of Oxytropis sericea were identified: one that accumulated high concentrations of swainsonine and another where swainsonine was not detected, or concentrations were near the detection threshold. The plants with high swainsonine concentrations had quantitatively higher amounts of endophyte. Alternatively, plants with low or no swainsonine detected had quantitatively lower endophyte amounts. In addition, swainsonine and endophyte concentrations were not distributed uniformly within the same plant when separated into stalks (leaves, scape(s), and flowers/pods). These findings provide evidence as to why plants in the same population accumulate different concentrations of swainsonine, and they have important implications for sampling of locoweed plants.

Detection of swainsonine-producing endophytes in Patagonian Astragalus species.

Swainsonine has been identified as the toxin in legumes belonging to the genera Astragalus and Oxytropis throughout the world including China, North America, and South America. Several South American Astragalus species have been reported to contain swainsonine; however, data is lacking to support the presence of a fungal symbiont in South American Astragalus species as has been shown for North American and Chinese Astragalus and Oxytropis species. The objective of this study was to investigate several South American species that have been reported to contain swainsonine for the presence of the fungal symbiont using culturing and PCR. Swainsonine was detected in field collections of A. pehuenches, A. illinii and A. chamissonis but not A. moyanoi, which is consistent with reports of toxicity regarding these species. The symbiont Alternaria section Undifilum was detected by PCR in all three species that contained swainsonine but not in A. moyanoi. A fungal symbiont was isolated from seeds of Astragalus pehuenches and A. illinii. The isolated symbiont from both respective species produced swainsonine in vitro, and was demonstrated to belong to the genus Alternaria section Undifilum by analysis of the nuclear ribosomal DNA. It is highly likely that Alternaria section Undifilum isolates will be associated with other South American Astragalus species that are reported to contain swainsonine.

Spontaneous outbreak of Astragalus pehuenches (Fabaceae) poisoning in cattle in Argentina.

This is the first report of a spontaneous outbreak of Astragalus pehuenches poisoning on a farm in Argentine Patagonia, where 63 out of 70 cattle died. The main clinical signs of affected animals were ataxia, balance loss and progressive emaciation. Purkinje cells presented vacuolation and marginalization of the nucleus. Astragalus pehuenches was detected in the paddock as well as in the ruminal content and fecal matter samples of the affected animals. Swainsonine concentrations in Astragalus specimens were found to be as high as 0.096%.

Assessment of the perinatal effects of maternal ingestion of Ipomoea carnea in rats.

It is believed that Ipomoea carnea toxicosis induces abnormal embryogenesis in livestock. Studies on rats treated with I. carnea aqueous fraction (AF) during gestation, revealed litters with decreased body weight, but the characteristic vacuolar lesions promoted by swainsonine, its main toxic principle, were observed only in young rats on postnatal day (PND) 7. However, these alterations could have resulted as consequence of swainsonine placental passage and/or damage or even ingestion of the contaminated milk by pups. Thus, this perinatal work was performed to verify the transplacental passage of swainsonine and its excretion into milk employing the cross-fostering (CF) procedure as a tool of study. Females were treated with AF or vehicle during gestation and after birth pups were fostered between treated and untreated dams. Pup body weight gain (BWG) and histopathology to observe vacuolar degeneration were performed on PND 3 and 7. In addition, swainsonine detection was performed in amniotic fluid and milk from rats treated with the AF during gestation or lactation. BWG was significantly lower only in pups from mothers treated with the plant and fostered to other treated mothers (AF-AF group of pups). The histopathology revealed that pups from treated mothers fostered to untreated ones showed the characteristic vacuolar lesions; however, the lesions from the AF-AF pups were more severe in both periods evaluated. Amniotic fluid and milk analysis revealed the presence of swainsonine excretion into these fluid compartments. Thus, the results from CF and the chemical analysis allowed concluding that swainsonine passes the placental barrier and affects fetal development and milk excretion participates in I. carnea perinatal toxicosis.

Screening for swainsonine among South American Astragalus species.

Swainsonine is a toxic alkaloid found in several plant genera worldwide. The objective of this study was to screen several South American Astragalus species for the toxin swainsonine. Swainsonine was detected in 16 of 30 Astragalus species using liquid and gas chromatography coupled with mass spectrometry. Information in regard to the swainsonine content of these species may provide important information on the risk of grazing these toxic species.

Intoxication by Ipomoea sericophylla and Ipomoea riedelii in goats in the state of Paraíba, Northeastern Brazil.

A disease of the nervous system was observed in goats from two farms of the semiarid of the state of Paraíba, northeastern Brazil. Ipomoea sericophylla was found in one farm and I. riedelii in the other. Both plants were administered experimentally to five goats each. Both plants induced clinical signs similar to those observed in spontaneous cases. Two goats died spontaneously and five were euthanatized. Three goats recovered after the withdrawal of the plants. Histological examination showed that all goats that died spontaneously or were euthanized had diffuse vacuolation of neurons, macrophages of lymphatic tissues, and epithelial cells of pancreas, thyroid, renal tubules and liver. On electron microscopy of Purkinje cells, numerous dilated membrane bordered vacuoles were identified as lysosomes. On lectin-histochemical analysis, cerebellar cells gave positive reactions to Concanavalia ensiformis, Triticum vulgaris, and succinylated-T. vulgaris, which indicate the storage of alpha-D-mannose, alpha-D-glucose, beta-D-N-acetyl-glucosamine, and acetyl-neuraminic acid. The chemical analysis of I. sericophylla and I. riedelii showed 0.11 and 0.14% of swainsonine, respectively. The latter also contained calystegines B1, B2 and C1. It is concluded that I. sericophylla and I. riedelli cause a lysosomal storage disease.

Maternal Ingestion of Ipomoea carnea: Effects on Goat-Kid Bonding and Behavior.

Ipomoea carnea is a toxic plant found in Brazil and other tropical and subtropical countries and often causes poisoning of livestock. The plant contains the alkaloids swainsonine and calystegines, which inhibit key cellular enzymes and cause systematic cell death. This study evaluated the behavioral effects of prenatal ingestion of this plant on dams and their kids. Twenty-four pregnant goats were randomly allocated into four treatment groups and received the following doses (g/kg BW) of fresh I. carnea: 0 (control group), 1.0 (IC1), 3.0 (IC3), and 5.0 (IC5) from day 27 of gestation until parturition. Dam and kid bonding and behavior were evaluated by several tests, immediately after birth until six weeks of age. Dams from IC3 and IC5 groups spent less time paying attention to the newborn. There was a lack of maternal-infant bonding due to I. carnea intoxication. Kids from treated dams had difficulty in standing, suckling, and in recognizing their mother hours after birth. I. carnea can also compromise the kids' ability to learn and to retain spatial memory. We suggest that kids from pregnant goats given I. carnea during gestation have significant behavioral alterations and developmental delays that may compromise their survival.

Analysis of Swainsonine and Swainsonine N-Oxide as Trimethylsilyl Derivatives by Liquid Chromatography-Mass Spectrometry and Their Relative Occurrence in Plants Toxic to Livestock.

There are limited data concerning the occurrence of swainsonine N-oxide in plants known to contain swainsonine and its relative impact on toxicity of the plant material. A liquid chromatography-mass spectrometry method based on a solvent partitioning extraction procedure followed by trimethylsilylation and analysis using reversed phase high-pressure liquid chromatography-mass spectrometry was developed for the analysis of swainsonine and its N-oxide. The concentrations of each were measured in several swainsonine-containing taxa as well as two endophytic isolates that produce swainsonine. In vegetative samples the relative percent of N-oxide to free base ranged from 0.9 to 18%. In seed samples the N-oxide to free base ratio ranged from 0 to 10%. The measured concentrations of swainsonine N-oxide relative to swainsonine only slightly increases the actual toxicity of the various plant samples in a combined assay of both compounds.

A swainsonine survey of North American Astragalus and Oxytropis taxa implicated as locoweeds.

Swainsonine, an indolizidine alkaloid with significant physiological activity, is an α-mannosidase and mannosidase II inhibitor that causes lysosomal storage disease and alters glycoprotein processing. Swainsonine is found in a number of plant species worldwide, and causes severe toxicosis in livestock grazing these plants, leading to a chronic wasting disease characterized by weight loss, depression, altered behavior, decreased libido, infertility, and death. Swainsonine has been detected in 19 Astragalus and 2 Oxytropis species in North America by thin layer chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry and a jack bean α-mannosidase inhibition assay. In addition, 5 species in North America are presumed to contain swainsonine based upon reports from field cases. Many of these plant species have not been analyzed for swainsonine using modern instrumentation such as gas or liquid chromatography coupled with mass spectrometry. To provide clarification, 22 Astragalus species representing 93 taxa and 4 Oxytropis species representing 18 taxa were screened for swainsonine using both liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Swainsonine was detected in 48 Astragalus taxa representing 13 species and 5 Oxytropis taxa representing 4 species. Forty of the fifty-three swainsonine-positive taxa had not been determined to contain swainsonine previously using liquid or gas chromatography coupled with mass spectrometry. The list of swainsonine-containing taxa reported here will serve as a reference for risk assessment and diagnostic purposes.