Locoweed Species Research Articles

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.

Water-Deficit Stress Tolerance Differs between Two Locoweed Genera (Astragalus and Oxytropis) with Fungal Endophytes

Locoweeds are plants of the genera Astragalus and Oxytropis (Fabaceae family) and are toxic to cattle, sheep, and horses. The toxic property of locoweeds is due to the alkaloid swainsonine (SWA), which is synthesized by an endophytic fungus Alternaria spp. section Undifilum. Although the endophyte–locoweed complex is often considered mutualistic, empirical evidence for benefits to host plants is lacking. This study: 1) compared the growth, photosynthesis, and leaf pigment and antioxidant concentrations between endophyte-infected and endophyte-free plants under well-watered and water-deficit conditions; and 2) measured SWA to determine whether SWA concentrations are attenuated by water deficit and leaf age. Locoweed species in this study were woolly loco and silky crazyweed. Endophyte-infected and endophyte-free (by removal of seed coat) seedlings, as confirmed by DNA analyses, were grown under greenhouse conditions for 6 mo, after which plants were subjected to three 12- to 15-d water-deficit periods that created sublethal drought conditions. Results suggest that the endophyte did not influence photosynthetic gas exchange and leaf pigment concentrations. Under well-watered conditions only, endophyte-infected woolly loco plants had lower shoot and root biomass and higher concentrations of α-tocopherol than endophyte-free plants. SWA analyses revealed taxon-specific effects of water deficit, with water deficit increasing SWA concentrations in young leaves of woolly loco but not affecting SWA concentration in silky crazyweed. These results suggest that the endophyte behaves as a parasite in woolly loco plants grown under optimal but not under water-limited conditions. Further, results indicate that drought conditions elevate the toxicity of woolly loco plants. Improved understanding of endophyte-locoweed interactions and factors influencing SWA levels will contribute to the development of livestock management strategies to predict toxicity in particular locoweed populations.

English

Leguminous locoweeds cause toxicosis to grazing animals in western China and western USA. Swainsonine, a toxic alkaloid, is produced by the endophytic fungus Alternaria section Undifilum sp. living within the locoweed plants. Nothing is known of the other endogenous fungi associated with locoweed and it is unknown if the presence of Alternaria sect. Undifilum sp., a potential mutualist, in a locoweed influences the fungal microbiome associated with the plant. To help address these questions, endogenous fungi associated with three locoweed species (Oxytropis glabra, Sphaerophysa salsula, and Astragalus variabilis) collected from grasslands from western China were evaluated. Fungi were isolated from the tissues and identified by morphological features and sequencing of the internal transcribed spacer (ITS) regions. A total of 1209 fungal isolates were obtained from 1819 tissues for an isolation rate of 66.5%. Alternaria sect. Undifilum oxytropis, Alternaria spp. and Fusarium spp. were most commonly isolated. Plant host species, plant part, and environment influenced the endogenous fungal communities isolated from the locoweed plants. There were significant differences in the diversity of fungal species isolated from O. glabra from two sites, and no differences between the diversity of fungi isolated from A. variabilis from two sites. Alternaria sect. Undifilum was found most frequently associated with toxic locoweeds. Plants or plant parts that did not yield this endophyte had more plant pathogenic fungi associated with them. This is the first report of the diversity of fungi associated with these locoweeds and the first to suggest a beneficial role for Undifilum. Key words: Fungal endophytes, Undifilum sp., locoweeds.

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.

De novo Transcriptome Assembly of a Chinese Locoweed (Oxytropis ochrocephala) Species Provides Insights into Genes Associated with Drought, Salinity, and Cold Tolerance

Background: Locoweeds (toxic Oxytropis and Astraglus species), containing the toxic agent swainsonine, pose serious threats to animal husbandry on grasslands in both China and the US. Some locoweeds have evolved adaptations in order to resist various stress conditions such as drought, salt and cold. As a result they replace other plants in their communities and become an ecological problem. Currently very limited genetic information of locoweeds is available and this hinders our understanding in the molecular basis of their environmental plasticity, and the interaction between locoweeds and their symbiotic swainsonine producing endophytes. Next-generation sequencing provides a means of obtaining transcriptomic sequences in a timely manner, which is particularly useful for non-model plants. In this study, we performed transcriptome sequencing of Oxytropis ochrocephala plants followed by a de nove assembly. Our primary aim was to provide an enriched pool of genetic sequences of an Oxytropis sp. for further locoweed research.Results: Transcriptomes of four different O. ochrocephala samples, from control (CK) plants, and those that had experienced either drought (20% PEG), salt (150 mM NaCl) or cold (4°C) stress were sequenced using an Illumina Hiseq 2000 platform. From 232,209,506 clean reads 23,220,950,600 (~23 G nucleotides), 182,430 transcripts and 88,942 unigenes were retrieved, with an N50 value of 1237. Differential expression analysis revealed putative genes encoding heat shock proteins (HSPs) and late embryogenesis abundant (LEA) proteins, enzymes in secondary metabolite and plant hormone biosyntheses, and transcription factors which are involved in stress tolerance in O. ochrocephala. In order to validate our sequencing results, we further analyzed the expression profiles of nine genes by quantitative real-time PCR. Finally, we discuss the possible mechanism of O. ochrocephala's adaptations to stress environment.Conclusion: Our transcriptome sequencing data present useful genetic information of a locoweed species. This genetic information will underpin further research in elucidating the environmental acclimation mechanism in locoweeds and the endophyte-plant association.

Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments.

Background: Oxytropis ochrocephala Bunge, an indigenous locoweed species in China, poses great threats to livestock on grasslands. There is a need for further genetic study in the plants per se, for understanding the basis of its acclimation mechanism in various unfavorable environmental conditions and to implement effective control measures. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method for gene expression analysis. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable reference genes is required. The aim of this study was to select the most stable reference genes for transcriptional analysis in O. ochrocephala.Results: We selected 12 candidate reference genes, 18S ribosomal RNA (18S RNA), actin2/7 (ACT7), β-actin (ACTB), actin101 (ACT101), actin11 (ACT11), β-tubulin (TUB), α-tubulin (TUA), glyceraldehyde-3-phosphate dehydrogenase-1 (GAPDH1), GAPDH2, metallothionein-like protein (MET), fructose-bisphosphate aldolase (FBA) and histone H3 (HIS), from the transcriptome datasets of O. ochrocephala and determined the suitability by analyzing their expression levels when exposed to a range of abiotic stress conditions. By employing software packages including geNorm, NormFinder and BestKeeper, HIS, ACT7, and ACT101 were assessed as the most suitable set for normalization in all samples. When normalized with the most stable reference genes, the expression patterns of the three target genes were in accordance with those in the transcriptome data, indicating that the reference genes selected in this study are suitable.Conclusions: The study provided appropriate reference genes for accurate normalization in qRT-PCR analysis in O. ochrocephala and emphasized the importance of validating reference genes for gene expression analysis under specific experimental condition. The usage of inappropriate reference gene would cause misinterpretation.

Influence of Seed Endophyte Amounts on Swainsonine Concentrations in Astragalus and Oxytropis Locoweeds

Locoism is a toxic syndrome of livestock caused by the ingestion of a subset of legumes known as locoweeds endemic to arid and semiarid regions of the western United States. Locoweeds contain the toxic alkaloid swainsonine, which is produced by the endophytic fungi Undifilum species. Two chemotypes of plants can coexist within toxic populations of locoweeds: chemotype 1 plants are defined as individuals containing swainsonine concentrations greater than 0.01% and quantitatively greater amounts of Undifilum, while chemotype 2 plants are defined as individuals containing less than 0.01% swainsonine and quantitatively smaller amounts of Undifilum. To elucidate the mechanisms that govern chemotypes, the amount of Undifilum in seeds/embryos was manipulated, thus altering subsequent swainsonine concentrations in three locoweed species: Astragalus mollissimus, Astragalus lentiginosus, and Oxytropis sericea. Chemotype 1 seeds that were fungicide-treated or had the seed coat removed resulted in plants with swainsonine concentrations comparable to those in chemotype 2 plants. Conversely, embryos from seeds of chemotypes 1 and 2 that were inoculated with the endophyte resulted in plants with swainsonine concentrations comparable to those of chemotype 1 plants. This reproducible interconversion between the two swainsonine chemotypes suggests that the quantity of endophyte present in the seed at the time of germination is a key determinant of the eventual chemotype. Additionally, this is the first report of the inoculation of locoweeds with the endophyte Undifilum species.

Transmission of the locoweed endophyte to the next generation of plants

Abstract Locoweed species of the legume genera Astragalus and Oxytropis (Fabaceae) contain the toxic alkaloid swainsonine, and poison livestock in semi-arid regions throughout the world. Recently, the endophyte Undifilum oxytropis (previously characterized as Embellisia spp.) was found in species of Astragalus and Oxytropis , and was shown to produce swainsonine. A survey of major locoweed species was conducted throughout the western U.S. in 2005 to verify the presence of the endophyte and swainsonine. Seed from these plants was collected to verify the transmission of the endophyte to progeny. In study 1, seed from each parent plant from which the endophyte could be cultured was germinated and seedlings were grown for 4–6 months and then harvested. The presence and quantity of the endophyte was determined by real-time PCR, and swainsonine concentration by LC/MS. The endophyte was transmitted to nearly all of the progeny, however, it was extremely low ( −1 , and swainsonine concentration ranged from 0.21 % to 0.79 %. In study 2, seeds were taken from parent plants in the original 2005 survey from which the endophyte could not be cultured, though its presence was detected by PCR. The seeds were germinated and seedlings were grown for 4 months. The endophyte was detected by PCR in 94 % of the progeny, but the amounts were very low ( −1 ) and swainsonine was not detected in any of the samples. It appears that U. oxytropis has near perfect (approaching 100 %) vertical transmission to the next generation of seedlings. However, a small proportion of the progeny had low endophyte/low or no swainsonine, and this characteristic was inherited in the next generation.

Swainsonine-producing fungal endophytes from major locoweed species in China

Locoweeds including the toxic species of Astragalus spp and Oxytropis spp. are widely distributed in the western region of China and result in a chronic neurological disease known as locoism in animals. To determine the presence of swainsonine-producing fungal endophyte of major locoweed species in China, endophytes were isolated from 8 locoweed species that including A. variabilis, A. strictus, O. glacialis, O. kansuensis, O. ochrocepala, O. sericopetala, O. glabra and O. latibracteata. Seven species of locoweed were confirmed contain substantial amounts of swainsonine and infect swainsonine-producing fungal endophyte. These endophytes were classified as Undifilim oxytropis according to the fungal morphology and phylogenetic analysis based on sufficient ITS sequences. PCR-RFLP analysis of IGS region showed that the interspecific or intraspecific variations were present among the endophytes from different locoweed species.

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.

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.

Effect of previous locoweed (Astragalus and Oxytropis species) intoxication on conditioned taste aversions in horses and sheep1

Locoweed species (Astragalus and Oxytropis spp.) are a serious toxic plant problem for grazing livestock. Horses and sheep have been conditioned to avoid eating locoweed using the aversive agent LiCl. The objective of this study was to determine if previous locoweed intoxication affects food aversion learning in horses and sheep. Horses and sheep were divided into 3 treatment groups: control (not fed locoweed and not averted to a novel feed); locoweed-novel feed averted (fed locoweed and averted to a novel feed); and averted (not fed locoweed and averted to a novel feed). Animals in the locoweed-novel feed averted groups were fed locoweed during 2 periods of 21 and 14 d, respectively, with each feeding period followed by a 14-d recovery period. Animals were averted to a novel test feed at the end of the first locoweed-feeding period, and periodically evaluated for the strength and persistence of the aversion. During the first recovery period, locoweed-novel feed averted horses ate less (9.5% of amount offered) of the test feed than did control horses (99.8%) and did not generally differ from averted horses (0%). During recovery period 2, locoweed-novel feed averted horses (4.3%) differed (P = 0.001) in consumption (% of offered) of the test feed from controls (100%) and the averted group (0%). Locoweed-novel feed averted sheep differed (P = 0.001) from controls (14.4 vs. 99.5%, respectively, during recovery period 1), whereas locoweed-novel feed averted sheep did not differ (P > 0.50) from averted sheep (0.6%). During the second recovery period, control sheep (100%) differed (P < 0.05) from averted (0%) and locoweed-novel feed averted (12.2%) groups. Two intoxicated sheep (locoweed-novel feed averted) partially extinguished the aversion during the first recovery period, but an additional dose of LiCl restored the aversion. Two of 3 intoxicated horses had strong aversions that persisted without extinction; 1 horse in the locoweed-novel feed averted group had a weaker aversion. These findings suggest that horses and sheep previously intoxicated by locoweeds can form strong and persistent aversions to a novel feed, but in some animals, those aversions may not be as strong as in animals that were never intoxicated.

Phylogenetic relationships among New MexicoAstragalus mollissimusvarieties andOxytropisspecies by restriction fragment analysis

The phylogenetic relationships within two major locoweed genera,AstragalusandOxytropis, and among varieties of woolly loco found in New Mexico were analyzed by comparing their chloroplastrpoC1 andrpoC2 gene sequences. Nucleic acids from locoweed species and varieties collected from different geographical locations in New Mexico were amplified using specific primer sets and subjected to restriction fragment analyses. Identity of the amplicons was confirmed by determining the 5′-end sequences from pea and woolly loco var.matthewsii. The amplified sequences from all samples were digested with 16 different restriction enzymes. Presence or absence of individual restriction fragments was scored as binary characters and used to develop a similarity coefficient matrix for cladistic analyses to determine the phylogenetic relationships. The target sequence was conserved, yielding 7% polymorphic data.Oxytropisspecies were monophyletic and, as expected, formed a clade distinct fromAstragalus. The average similarity coefficient among woolly loco varieties was very high (0.9733), but the varieties still separated into three different clades. The phylogenetic relationship among woolly loco varieties coincided with their geographic distribution but was unrelated to insect feeding preference.

Uptake, Translocation, and Metabolism of Picloram and Metsulfuron Methyl by Two Locoweed Species

The locoweeds, woolly loco and silky crazyweed, contribute to livestock poisoning in the western United States. Differences in response of these two locoweeds to foliar-applied picloram and metsulfuron was investigated by evaluating differences in herbicide uptake, translocation, and metabolism. Silky crazyweed compared to woolly loco was more than 10 times as sensitive to increasing rates of either herbicide. The two species absorbed 8 to 15%, and 11 to 17% of applied picloram and metsulfuron, respectively. Translocation of picloram and metsulfuron out of treated leaflets of either species was less than 3% of that absorbed after 96 h. Approximately 70 and 100% of the absorbed herbicides remained as picloram and metsulfuron, respectively, in both species. Therefore, differences in picloram or metsulfuron uptake, translocation, and metabolism appear inadequate to account for the differential response to each herbicide by the two locoweed species. Selectivity differences between these locoweed genera to picloram and metsulfuron most likely are due to sensitivity differences at sites of action.

Seasonal Grazing of Locoweeds by Cattle in Northeastern New Mexico

Locoweed poisoning generally occurs in early spring. We evaluated cattle grazing of woolly locoweed (Astragalus mollissimus var. mollissimus Torr.) at Gladstone, N.M., and of white locoweed (Oxytropis sericea Nutt. ex T&G) at Capulin, N.M., through the spring and into early summer as the phenological development of warm-season grasses progressed from dormancy to rapid growth. Diets of 8 mature cows were quantified by bite count at each location. Cattle initially rejected woolly locoweed at Gladstone, even though it was the only green forage available in late March and early April. Gladstone cattle were then restricted to a small 7-ha pasture where high grazing pressure and limited feed forced them to graze woolly locoweed (41% of bites). When these cows returned to a larger pasture of unlimited forage availability, they continued eating woolly locoweed (23% of bites). At Capulin, cattle with a history of eating locoweed (loco-eaters) consumed more white locoweed (23% of bites) thin cattle without a history of eating locoweed (6% of bites) during the April grazing period. When warm-season grasses started rapid growth and locoweed matured in June, cattle ceased grazing both locoweed species.

Utilization of White Locoweed (Oxytropis sericea Nutt.) by Range Cattle

Utilization studies conducted on a high mountain range determined the quantity and timing of white locoweed (Oxytropis sericeu Nutt.) consumption by cattle. Paired plots (one caged and one grazed) were clipped at the end of the grazing season to determine seasonal utilization. Biweekly visual appraisals were used to estimate percentage leaf grazed and number of reproductive stalks grazed and thus determine utilization patterns as the season progressed. Loco comprised 26% of the standing crop. Thirty-four percent of the available loco was utilized during the grazing season. Loco flower and pods (heads) were preferred to leaves. Utilization of loco heads increased linearly as the season progressed. LOCO leaves were not consumed until the last 3 weeks of the grazing season. Loco heads also contained the highest concentration of the toxic alkaloid, swainsonhre. Locoweed poisoning of livestock has been the most widespread poisonous plant problem in the western United States (James et al. 1981). Marsh (1909) reviewed reports of locoweed poisoning dating back to 1873 and conducted feeding trials that showed conclusively that white locoweed (Astragalus lambertiinow considered to be Oxytropis sericea Nutt.) and wooly loco (A. mollissimus Torr.) caused locoism in cattle, sheep, and horses. Mathews (1932) conducted further studies to determine quantities of Earle loco (A. mollissimus var earlei Green ex Rydb.) and Garbancillo (A. wootoni Sheld.) required to produce clinical signs of toxicity in domestic livestock. The toxin in locoweeds has been identified as the indolizidine alkaloid swainsonine (Molyneux and James 1982). Desert and semidesert locoweed species (A. lentiginosus Dougl., A. pubentissimus Torr. and Gray, A. mollissimus Torr.) characteristically germinate and become established during a warm, wet fall, remain green over winter, and accelerate growth in early spring. Most livestock losses occur during the winter and early spring when other forage is scarce, or the existing forage is dry and the Authors are with USDA, Agricultural Research Service, Poisonous Plant Research Laboratory, 115OEast 14OONorth, Logan, Utah8432l;and withTexasTechUniversity, Lubbock. The assistance of Steve Fitzwater for his assistance in data collection and Russ Molyneux for alkaloid determination is gratefully acknowledged. This is Journal Paper Number 3103, Utah Agr. Exp. Sta. Manuscript accepted 21 October 1985. 344 locoweed is green (James et al. 1968, James et al. 1969). Due to the habituating properties of loco, livestock that begin eating it will continue to consume it even when good quality green feed is available (Peters and Sturdevent 1908, Marsh 1909, James et al. 1969). White locoweed differs somewhat from other locoweed species. It occurs on the western plains and foothills of the Rocky mountains and up to 3,353 m on some Intermountain ranges (Barneby 1952). It is palatable and livestock graze it throughout the growing season (Marsh 1909). Its population cycles on high mountain ranges apparently are not influenced as much by weather conditions as other locoweed species (James personal observation). Payne (1957) classified it as an increaser species. Recently it has been identified as a predisposing factor in high mountain brisket disease, or congestive right-sided heart failure, in cattle (James et al. 1983). Economic loss occurs annually from the interaction of these two diseases (Barnard 1984). Little is known about why livestock graze locoweeds. Marsh (1909) made some astute observations of livestock grazing white locoweed and made some broad general recommendations to reduce losses (Marsh 1913). However, until we know when cattle eat white locoweed, how much is required to produce intoxication, and what environmental or management factors influence consumption, little can be done to reduce losses other than keeping livestock off infested ranges. The purpose of this study was to describe utilization patterns of white locoweed by cattle on a high mountain range and identify factors that may influence consumption. Once we understand the conditions under which cattle consume white locoweed, management strategies can be developed to reduce losses.