Swainsonine Production Research Articles

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 β-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.

Screening of Endophytic Fungi in Locoweed Induced by Heavy-Ion Irradiation and Study on Swainsonine Biosynthesis Pathway.

Swainsonine (SW) is a substance with both animal neurotoxicity and natural anticancer activity produced by the metabolism of endophytic fungus Alternaria section Undifilum oxytropis of locoweed. This paper produced SW by fermentation of the endophytic fungus A. oxytropis of locoweed and obtained the optimal ultrasonic-assisted extraction process of SW by the response surface methodology. Meanwhile, four mutant strains with significant and stable SW-producing properties were screened out after the mutagenesis of A. oxytropis by heavy-ion irradiation. Of these, three were high-yielding stains and one was a low-yielding strain. In addition, through the analysis of metabolomics studies, it was speculated that the different SW production performance of the mutant might be related to the biosynthesis and utilization of L-lysine, L-2-aminoadipate-6-semialdehyde, etc. These results laid the foundation for the expansion of SW production, artificial construction of low-toxic locoweed and clarification of the SW biosynthesis pathway in A. oxytropis.

Host-Species Variation and Environment Influence Endophyte Symbiosis and Mycotoxin Levels in Chinese Oxytropis Species.

Oxytropis plants are widely distributed in the grasslands in northern China. Some Oxytropis species have been reported to contain the mycotoxin swainsonine, an alkaloid which causes poisoning in livestock, referred to as locoism. Previous studies showed that endophytic fungi (Alternaria oxytropis) symbiotically associate with these Oxytropis species to produce swainsonine. However, the influence of variation within the Oxytropis genus on the fixation or loss of symbiosis and toxicity is poorly understood, as is the influence of environmental factors. Here we used a collection of 17 common Oxytropis species sampled in northern China to assess genetic diversity using genotyping by sequencing which was compared with the levels of the endophyte and swainsonine. Results showed that nine Oxytropis species have detectable A. oxytropis colonisation, and seven Oxytropis species contain sufficient swainsonine to be considered poisonous, whereas the rest may be non-toxic. Species variation rather than the genetic lineage was associated with the fixation or loss of endophyte and swainsonine production, which appears to have resulted from genetic drift. Genotype × Environment (G × E) effects were also found to influence endophyte and swainsonine levels amongst species of the Oxytropis genus. Our study will provide a better understanding about the evolutionary basis of A. oxytropis symbiosis and swainsonine biosynthesis in locoweeds.

Swainsonine producing performance of Alternaria oxytropis was improved by heavy-ion mutagenesis technology.

Swainsonine, an indolizidine alkaloid, is a promising anti-tumorigenic compound. Biological production of swainsonine was prospective, but the low swainsonine yield of wild type Alternaria oxytropis limited its production on a large scale. In present work, a stable A. oxytropis mutant UO1 with swanisonine yield of 14.84% higher than the wild-type strain was successfully obtained after heavy-ion irradiation. The A. oxytropis mutant UO1 and original wild-type strain were futher evaluated for SW concentrations under different factors. Results showed that the optimum culture temperature was 25°C. The optimum initial medium pH was 6.5 and the optimum inoculum size was 2mL per 200mL. Addition of the biosynthetic precursor L-pipecolic acids and L-lysine appropriately could increase the SW synthesis. These findings provided a theoretical basis and scientific data for the industrial production of swainsonine.

Unveiling of Swainsonine Biosynthesis via a Multibranched Pathway in Fungi.

The indolizidine alkaloid swainsonine (SW) is a deadly mycotoxin to livestock that can be produced by different plant-associated fungi, including the endophytic entomopathogenic fungi Metarhizium species. The SW biosynthetic gene cluster has been identified but the genetic mechanism of SW biosynthesis remains obscure. To unveil the SW biosynthetic pathway, we performed gene deletions in M. robertsii, heterologous expression of a core biosynthetic gene, substrate feedings, mass spectrometry, and bioassay analyses in this study. It was unveiled that SW is produced via a multibranched pathway by the hybrid nonribosomal peptide-polyketide synthase (NRPS-PKS) gene cluster in M. robertsii. The precursor pipecolic acid can be converted from lysine by both the SW biosynthetic cluster and the unclustered genes such as lysine cyclodeaminase. The hybrid NRPS-PKS enzyme produces three intermediates with and without domain skipping. Intriguingly, the biosynthetic process is coupled with the cis to trans nonenzymatic epimerization of C1-OH for both hydroxyl- and dihydroxyl-indolizidine intermediates. We also found that SW production was dispensable for fungal colonization of plants and infection of insect hosts. Functional characterization of the SW biosynthetic genes in this study may benefit the safe use of Metarhizium fungi as insect biocontrol agents and the management of livestock pastures from SW contamination by genetic manipulation of the toxin-producing fungi.

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), andglyceraldehyde-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.

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.

Clarification of Swainsonine Biosynthesis by a Multi-Branched Pathway and Non-Accumulation of Mycotoxin in Plants after Fungal Colonization

The indolizidine alkaloid swainsonine (SW) is a deadly mycotoxin to livestock. The genetic mechanism of SW biosynthesis remains obscure for decades. Here we report that SW is produced via a multi-branched pathway by a hybrid nonribosomal peptide/polyketide synthase (NRPS-PKS) gene cluster in the endophytic entomopathogenic fungus Metarhizium robertsii. The precursor pipecolic acid can be redundantly converted from lysine by both clustered and unclustered genes. The hybrid enzyme that begins with a NRPS module produces three intermediates with and without domain skipping. Intriguingly, the biosynthetic process is coupled with the trans to cis non-enzymatic epimerization of C1-OH for both hydroxyl- and dihydroxyl-indolizidine intermediates. We also found that SW production is dispensable for fungal colonization of plants and the infection of insect hosts. Our finding that SW does not accumulate in planta after Metarhizium colonization provides a notable indication of safety when using this fungus to control insect pests.

Locoweed Endophytes: A Review

Locoweeds, which are a common name for poisonous species of the genera Astragalus and Oxytropis, are toxic leguminous plants through a symbiotic association with the fungal endophyte Alternaria section Undifilum. The fungus produces an indolizidine alkaloid, swainsonine, which causes a toxicosis of grazing animals, named locoism. By what means stress from environment or genetics of locoweed endophytes affect fungal growth and synthesis of swainsonine as well as how fungal endophyte synthesize swainsonine in locoweeds, in addition to disruption of the toxic alkaloid swainsonine in locoweed plants, have not been well identified. To answer the above questions, further research is needed as locoweed plants are most palatable to grazing animals especially when locoweed plants are at flowering stage. This review outlines the present situation on locoweed plants in China, production of swainsonine, detection and interaction of locoweed endophytes as well as their transmission within locoweed plants .

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.

The toxicology mechanism of endophytic fungus and swainsonine in locoweed.

Locoweed is a perennial herbaceous plant included in Astragalus spp. and Oxytropis spp. that contains the toxic indolizidine alkaloid swainsonine. The livestock that consume locoweed can suffer from a type of toxicity called locoism. There are aliphaticnitro compounds, selenium, selenium compounds, and alkaloids in locoweed. The toxic component in locoweed has been identified as swainsonine, an indolizidine alkaloid. Swainsonine inhibits lysosomal a-mannosidase and mannosidase II, resulting in altered oligosaccharide degradation and incomplete glycoprotein processing. Corresponding studies on endophytic fungi producing swainsonine have been isolated from a variety of locoweed, and these endophytic fungi and locoweed have a close relationship. Endophytic fungi can promote the growth of locoweed and increase swainsonine production. As a result, livestock that consume locoweed exhibit several symptoms, including dispirited behavior, staggering gait, chromatopsia, trembling, ataxia, and cellular vacuolar degeneration of most tissues by pathological observation. Locoism results in significant annual economic losses. Therefore, in this paper, we review the current research on locoweed, including that on locoweed species distribution in China, endophyte fungus in locoweed, the toxicology mechanism of locoweed, and the swainsonine effect on reproduction.

Swainsonine, a novel fungal metabolite: optimization of fermentative production and bioreactor operations using evolutionary programming

The optimization of bioreactor operations towards swainsonine production was performed using an artificial neural network coupled evolutionary program (EP)-based optimization algorithm fitted with experimental one-factor-at-a-time (OFAT) results. The effects of varying agitation (300-500 rpm) and aeration (0.5-2.0 vvm) rates for different incubation hours (72-108 h) were evaluated in bench top bioreactor. Prominent scale-up parameters, gassed power per unit volume (P g/V L, W/m(3)) and volumetric oxygen mass transfer coefficient (K L a, s(-1)) were correlated with optimized conditions. A maximum of 6.59 ± 0.10 μg/mL of swainsonine production was observed at 400 rpm-1.5 vvm at 84 h in OFAT experiments with corresponding P g/VL and K L a values of 91.66 W/m(3) and 341.48 × 10(-4) s(-1), respectively. The EP optimization algorithm predicted a maximum of 10.08 μg/mL of swainsonine at 325.47 rpm, 1.99 vvm and 80.75 h against the experimental production of 7.93 ± 0.52 μg/mL at constant K L a (349.25 × 10(-4) s(-1)) and significantly reduced P g/V L (33.33 W/m(3)) drawn by the impellers.

Real encoded genetic algorithm and response surface methodology to optimize production of an indolizidine alkaloid, swainsonine, from Metarhizium anisopliae

Response surface methodology (RSM) and artificial neural network-real encoded genetic algorithm (ANN-REGA) were employed to develop a process for fermentative swainsonine production from Metarhizium anisopliae (ARSEF 1724). The effect of finally screened process variables viz. inoculum size, oatmeal extract, glucose, and CaCl2 were investigated through central composite design and were further utilized for training sets in ANN with training and test R values of 0.99 and 0.94, respectively. ANN-REGA was finally employed to simulate the predictive swainsonine production with best evolved media composition. ANN-REGA predicted a more precise fermentation model with 103 % (shake flask) increase in alkaloid production compared to 75.62 % (shake flask) obtained with RSM model upon validation.

Detection of swainsonine and isolation of the endophyte Undifilum from the major locoweeds in Inner Mongolia

Locoweeds are Astragalus and Oxytropis species that contain the toxic alkaloid swainsonine, causing widespread poisoning of livestock in Inner Mongolia. Taxa (Astragalus, Oxytropis, Sphaerophysa, and Sophora species) suspected of causing locoism and/or poisoning in Inner Mongolia were surveyed for swainsonine and Undifilum, the fungal endophyte responsible for the production of swainsonine. Swainsonine was detected at concentrations greater than 0.01% in Astragalus variabilis and Oxytropis glabra. The endophyte Undifilum was detected by culturing and PCR in samples containing swainsonine concentrations greater than 0.01%. In some specimens of A. variabilis and O. glabra swainsonine was not detected or concentrations were less than 0.01%. In these samples the endophyte could not be cultured, but was detected by PCR. Additionally, contrary to previous reports the quinolizidine alkaloids, thermopsine, anagyrine, and lupanine, were not detected in O. glabra and Oxytropis ochrocephala, however the quinolizidine alkaloids, sophoridine, sophocarpine, and sophoramine were detected in Sophora alopecuroides as previously reported.

Optimization of different process variables for the production of an indolizidine alkaloid, swainsonine from Metarhizium anisopliae

Swainsonine is a polyhydroxylated indolizidine alkaloid having anticancer, antimetastatic, antiproliferative and immunomodulatory activities and also potential therapeutic applications against AIDS. In the present study, ten isolates of M. anisopliae were screened and enzyme assayed for the production of swainsonine in different media (Complex oatmeal, Czapekdox media with and without lysine (8% w/v) and Sabouraud dextrose broth (SDB)). Among these strains, ARSEF 1724 (UM8) was found to produce highest amount of swainsonine (1.34 μg/l) after 72 h of incubation under shake flask conditions at 180 rpm and 28 °C in complex oatmeal media. In order to maximize the yield of swainsonine the media composition including macro and micronutrients were optimized. The process variables including the chemical factors like carbon sources, nitrogen sources of both organic and inorganic nature and pH with constant inoculum size (1 × 10(8) spores/ml) were screened using classical one-factor-at-a-time (OFAT) approach to find their optimum levels. The present study shows that the nutrient requirement is specific for each strain of Metarhizium. Oatmeal extract (6%) was found to be the best supporting media along with nitrogen source, glucose (2%) as best carbon source and pH (~5) as the best for swainsonine production.

Quantitative PCR Method To Measure the Fungal Endophyte in Locoweeds

A fungal endophyte ( Undifilum oxytropis ) has been implicated in the synthesis of swainsonine in Oxytropis and Astragalus species, commonly known as locoweeds. A quantitative PCR method has been developed to measure the amount of endophyte in Oxytropis and Astragalus species. The limit of quantitation was estimated to be 0.2 pg of endophyte/ng of total DNA. This method of analysis was used to quantify the amount of endophyte in 10 plants each of Oxytropis sericea (white point locoweed), Astragalus mollissimus (wooly locoweed), and Astragalus lentiginosus (spotted locoweed). A significant amount of individual plant variability was observed in endophyte content among individuals in all three species. In one O. sericea and one A. lentiginosus plant swainsonine concentrations were near or below the limit of detection. These plants also had the lowest amounts of endophyte when compared to the other specimens. This method will be a useful tool in further investigating the role the endophyte plays in swainsonine production in various locoweed species.

Undifilum, a new genus for endophyticEmbellisia oxytropisand parasiticHelminthosporium bornmuellerion legumes

Fungal endophytes of Oxytropis kansuensis Bunge from China, previously described as Embellisia oxytropis Q. Wang, Nagao & Kakish, and endophytes of Oxytropis sericea Nutt. and Oxytropis lambertii Pursh from the United States were compared and are reported here as conspecific members of a new genus in the Pleosporaceae, Undifilum, based on morphological and molecular analyses. Morphological comparisons revealed characters that are similar to those of the genus Embellisia including conidia ovate to obclavate to long ellipsoid, straight or slightly to decidedly inequilateral with occasionally one or two cells distinctly swollen, and transepta occasionally thickened, dark, and rigid in comparison with the exterior conidium wall. However, upon germination, conidia produced unique and diagnostic germ tubes that were wavy or undulating in their growth until branching. Moreover, all isolates were found to produce the toxic alkaloid swainsonine. Parsimony analysis of sequences from ITS1–5.8S–ITS2, glyceraldehyde-3-phosphate dehydrogenase gene, and mitochondrial small subunit rDNA data sets revealed that the Oxytropis endophytes formed a clade distinct from other Embellisia species and species in the genera Alternaria , Ulocladium , Nimbya , and Crivellia . A second taxon, Helminthosporium bornmuelleri P. Magnus, was reexamined and found to possess similar morphological features to those of the Oxytropis isolates, but lacked swainsonine production. Sequence analysis placed this second taxon in the same clade with high bootstrap support. The distinct morphology and genetics of these taxa demonstrates that these fungi, both recovered from legumes, represent a new genus, hereinafter described as Undifilum . The two species now placed in this genus are redescribed as Undifilium oxytropis and Undifilium bornmuelleri .

WHITE LOCOWEED TOXICITY IS FACILITATED BY A FUNGAL ENDOPHYTE AND NITROGEN-FIXING BACTERIA

Mutualistic interactions with fungal endophytes and dinitrogen-fixing bacteria are known to exert key biological influences on the host plant. The influence of a fungal endophyte alkaloid on the toxicity of a plant has been documented in Oxytropis sericea. Oxytropis sericea is a perennial legume responsible for livestock poisoning in western North America. Livestock poisoning is attributed to the alkaloid swainsonine, which is synthesized inside the plant by the fungal endophyte Embellisia sp. In this study, the ability of Oxytropis sericea to form a dinitrogen-fixing symbiosis with Rhizobium and the effects of this symbiosis on the production of swainsonine by Embellisia sp. were evaluated in a greenhouse environment. Seeds of O. sericea were grown in plastic containers. Twenty-week-old O. sericea seedlings were inoculated with four strains of Rhizobium. Twenty weeks after inoculation, plant growth and root nodulation by Rhizobium were measured. Dinitrogen fixation was confirmed using an acetylene reduction assay (ARA) on excised root nodules. Dry leaves were analyzed for swainsonine content. A second set of plants was treated with fungicide to evaluate the effect of reduced fungal endophyte infection on plant growth and swainsonine production. All inoculated plants produced indeterminate nodules. The ARA indicated that 98% of the excised nodules were fixing dinitrogen. Rhizobium-treated plants had greater swainsonine concentrations than the non-inoculated controls. Plants that established from seeds treated with fungicide had lower biomass than non-fungicide-treated controls and plants treated with foliar fungicide. Plants treated with foliar fungicide and the controls had greater swainsonine concentrations than the plants that received seed fungicide. This greenhouse study is the first report of nodulation and dinitrogen fixation in O. sericea. It also demonstrates that dinitrogen fixation increases the production of swainsonine in O. sericea plants infected with Embellisia sp. Results from this study suggest that dinitrogen fixation affects swainsonine production and has the potential to support the symbiosis between Embellisia sp. and O. sericea when soil nitrogen is limited. Oxytropis sericea competitiveness appears to be facilitated by an ability to simultaneously associate with Rhizobium and a fungal symbiont.

Production of swainsonine by fungal endophytes of locoweed

Consumption of locoweeds, legumes endemic in arid western USA, has long been associated with locoism, a disease of ruminant animals. To explore the relationship between fungi associated with locoweed and locoweed toxicity, 11 locoweed populations from various sites in New Mexico were assessed for endophytic fungi. Endophytes were isolated from the leaves, stems, seeds, and flowers of eight populations of the toxic locoweeds Astragalus mollissimus, Oxytropis lambertii, and O. sericea. Fungal cultures grew very slowly and sporadically produced subcylindrical conidia with very dark transverse septa. All cultured endophytes produced the alkaloid swainsonine, which causes locoism. Endophyte-infected locoweed populations produced swainsonine, and the swainsonine level of endophyte strains in vitro was highly correlated with the swainsonine level of their host plant populations. The rDNA ITS from mycelia from four endophyte isolates and beta-tubulin encoding regions from mycelia of 18 fungal endophyte isolates were amplified using PCR and the nucleic acid sequences were analyzed. The nucleic acid sequences of the beta-tubulin encoding regions were essentially identical among all the endophytes regardless of plant genus and locations. Morphological evidence and sequence analysis of the ITS region suggest that the endophytes are most closely related to Embellisia. However, with the paucity of Embellisia species represented in sequence databases, precise taxonomic placement will await further study.

Optimization of agitation for production of swainsonine from Metarhizium anisopliae in stirred tank and airlift reactors

The optimal agitation rate for production of swainsonine from Metarhizium anisopliae grown in batch stirred tank reactors (2 to 20 l) was 400 rpm with a mixed hyphal and pelleted morphology where the specific swainsonine production rate was 9×10−2 mg g−1 cell dry wt h from 87 to 142 h. Culture of the fungus in a 6-l airlift reactor produced ‘loose’ pellets and the production of swainsonine started at least 24 h earlier than in the stirred tank reactor. The final yield (5.9 mg swainsonine g−1 cell dry wt) after 168 h in the airlift reactor was 18% less than those obtained in the stirred tank reactor with an agitation rate of 400 rpm.