Note pubs.acs.org/jnp
Production of the Alkaloid Swainsonine by a Fungal Endophyte in the Host Swainsona canescens Daniel S. Grum,† Daniel Cook,*,† Deana Baucom,‡ Ivan W. Mott,§ Dale R. Gardner,† Rebecca Creamer,‡ and Jeremy G. Allen⊥ †
USDA/ARS Poisonous Plant Research Laboratory, 1150 East 1400 North, Logan, Utah 84341, United States Department of Entomology, Plant Pathology, and Weed Science, New Mexico State University, Las Cruces, New Mexico 88001, United States § USDA/ARS Forage and Range Research Laboratory, Logan, Utah 84322, United States ⊥ Animal Health Laboratories, Department of Agriculture and Food, 3 Baron-Hay Court, South Perth WA 6151, Australia ‡
S Supporting Information *
ABSTRACT: 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 Undif ilum 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 Undif ilum species using morphological and phylogenetic analyses.
T
oxic groups of legumes belonging to the Astragalus, Oxytropis, and Swainsona genera have been noted by ranchers in the Americas, Asia, and Australia.1−5 Consumption of some Astragalus and Oxytropis species by livestock in the Americas results in a condition known as “locoism”, while consumption of some Swainsona species in Australia causes livestock, most commonly sheep, to become “peastruck”.1−3 Clinical signs and pathology are similar in animals intoxicated by locoweed species and Swainsona species.6 Consumption of these plants by grazing animals leads to a chronic disease characterized by weight loss, depression, altered behavior, decreased libido, infertility, and death, resulting in significant economic losses on an annual basis.7 Swainsonine (Figure 1), a toxic indolizidine alkaloid, was first identified in the Australian legume Swainsona canescens.8 Subsequently swainsonine was
identified as the toxin in Astragalus and Oxytropis species that cause locoism.9 As well as occurring sporadically in the Fabaceae, swainsonine has been reported in two other plant families. Ipomoea carnea and Turbina cordata,10,11 both members of the Convolvulaceae family, and Sida carpinofolia,12 a member of the Malvaceae family, are all reported to contain swainsonine. The sporadic occurrence of some natural products such as swainsonine in plants brings into question the origin and significance of these products as chemotaxonomic markers. Three possible mechanisms may explain their sporadic occurrence in unrelated taxa:13 first, the biosynthetic pathways of a given natural product may have evolved multiple times over evolutionary history; second, the genes responsible for the biosynthesis of a particular natural product may have been horizontally transferred between unrelated taxa;14 and third, the natural product may be produced by a microbe associated with the plant. Fungi including Rhizoctonia leguminicola (Ceratobasidiaceae) and Metarhizium anisopliae (Clavicipitaceae) are reported to produce swainsonine.15,16 Additionally, fungal endophytes of the genus Undif ilum (Pleosporaceae) found in the genera Astragalus and Oxytropis (Fabaceae),17−19 previously described
Figure 1. Structure of the indolizidine alkaloid swainsonine.
Received: April 1, 2013
© XXXX American Chemical Society and American Society of Pharmacognosy
A
dx.doi.org/10.1021/np400274n | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
as Embellesia species,20 have been reported to be responsible for the production of swainsonine.21 Endophytic Undif ilum species described to date are vertically transmitted, and removal of the seed coat or fungicide treatment of seeds results in plants with little or no detectable swainsonine.22−24 Two chemotypes of plants coexist within some Astragalus and Oxytropis populations: 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% (generally not detected) and quantitatively smaller amounts of Undif ilum.25,26 Endophyte amounts are correlated with concentrations of swainsonine in Astragalus and Oxytropis locoweeds.27,28 Swainsona is a large genus of the Fabaceae family with all species being endemic to Australia and New Zealand. The genus Swainsona is closely related to the Astragalus and Oxytropis genera of Asia and the Americas.29 Swainsoninecontaining Astragalus and Oxytropis species have consistently been found to be associated with fungal endophytes in the genus Undif ilum.18,30 It seems probable that a fungal endophyte may be associated with Swainsona taxa that contain swainsonine. Here we report the examination of the endemic Australian species, S. canescens, for a fungal endophyte that produces swainsonine. Two species of Swainsona, S. canescens, which has a history of poisoning sheep, and S. maccullochiana, which is anecdotally benign,31 were examined for the presence of swainsonine and an endophyte. Swainsonine was detected at a concentration of 0.6 mg/g in seeds of S. canescens, while it was not detected in S. maccullochiana seeds. A single fungal endophyte was successfully cultured from six of eight S. canescens seeds, while no fungal endophytes were cultured from the 10 S. maccullochiana seeds examined. A fungal endophyte was cultured from seven of eight S. canescens seed coats that were mechanically separated from the embryo, while no fungal endophyte was cultured from the eight embryos. These results, where an endophyte can be cultured from the seed coat but not the embryo, suggest that the endophyte resides primarily in the seed coat, which is consistent with reports in Astragalus and Oxytropis that contain swainsonine.22−24 The endophyte isolates from S. canescens all produced swainsonine in vitro as verified by GC-MS (Figure 2, Figure S1, Supporting Information)32 and LC-MS/MS (Figure S2, Supporting Information).33 Both the fungal biomass and the culture media contained swainsonine. The mean concentration of swainsonine for the endophytes from S. canescens was 7.4 mg/g, with a range of 3.7−11.9 mg/g (Table 1). Undifilum species from Astragalus and Oxytropis species of North America also produced swainsonine in vitro (Table 1). The fungal biomass and the culture media contained swainsonine in these isolates as well. The mean concentrations of swainsonine differed among Undifilum species from different hosts (p < 0.001). For example, U. cinereum from A. mollissimus had a mean concentration of 7.5 mg/g, U. f ulvum from A. lentiginosus had a mean concentration of 16 mg/g, and U. oxytropis from O. sericea had a mean concentration of 1.9 mg/g (Table 1). Interestingly, the rank order of the concentrations of swainsonine from the Astragalus and Oxytropis endophytes are similar to the concentrations found in each of the respective plant species.23 These results are also consistent with previous reports where in planta swainsonine concentrations correlated
Figure 2. GC-MS chromatogram and mass spectrum of swainsonine, as the tri-TMS derivative (tR = 14.54 min) and key fragment ions. The sample was from that produced from the endophyte of Swainsona canescens.
Table 1. Mean Swainsonine Production of Different Undif ilum Species Isolated from Astragalus, Oxytropis, and Swainsona Species plant species
fungal endophyte
mean swainsonine (mg/g)
range
SE
S. canescens A. mollissimus A. lentiginosus O. sericea
Undif ilum sp. U. cinereum U. f ulvum U. oxytropis
7.4a 7.5a 16a 1.9b
3.7−11.9 1.4−22.5 4.9−32.6 0.3−8.4
0.5 2.9 4.3 1.1
a,b
Different letters within column are statistically significant (p < 0.05).
with swainsonine concentrations of the corresponding endophyte isolated from the respective host.21,34 The endophyte isolates from S. canescens were identified as an Undif ilum species (Pleosporaceae) based on their morphology. The isolates were slow growing like other Undif ilum species (Figure S3, Supporting Information). Germinating conidia from the isolates produced the distinctive and diagnostic germ tubes that are characteristic of the genus Undif ilum, being wavy or undulating in their growth until branching (Figure 3).17 Furthermore, it was authenticated to be an Undifilum species by the molecular analysis of the internal transcribed spacer rDNA (ITS) (Figure S4, Supporting Information). All isolates of the endophyte yielded identical sequences of the ITS. The ITS sequences obtained from the three isolates of the endophyte have been deposited with GenBank (JX674068, JX674069, JX674070). The endophyte isolates from S. canescens and seed purchased from B&T World Seeds were deposited at the Fungal Collection of the Intermountain Herbarium at Utah State University (UTC 259540). Swainsonine concentrations in plants derived from seeds of S. canescens purchased from B&T World Seeds and from the two voucher specimens of S. canescens collected in Western Australia ranged from not detected to 3.7 mg/g, with a mean B
dx.doi.org/10.1021/np400274n | J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
endophyte detected by culturing or Undifilum detected by PCR. In conclusion, an endophyte was isolated from S. canescens that produces swainsonine in vitro. This endophyte belongs to the genus Undif ilum, but is morphologically and phylogenetically distinct from other Undif ilum species so far described. Furthermore the data support that this Undif ilum species is vertically transmitted. It seems highly probable that Undif ilum may be associated with other Swainsona species that are reported to contain swainsonine, such as S. galegifolia,31 and this merits further investigation. As swainsonine occurs sporadically in two other plant families, the Convolvulaceae and Malvaceae, it seems highly probable that a swainsonineproducing endophyte may be associated with these plants as well. In fact, a fungal endosymbiont belonging to the fungal order Chaetothyriales was recently isolated from Ipomoea carnea, which produces swainsonine in vitro.36
Figure 3. Wavy germ tube from germinating conidia from an endophytic isolate from Swainsona canescens on V8 agar,; scale bar = 10 μm.
concentration of 2 mg/g (Table 2). These highly variable concentrations of swainsonine are similar to observations in
■
Table 2. Swainsonine Concentrations and Endophyte Detection and Quantitation by PCR and Culturing in Swainsona canescens and Swainsona macculochiana
General Experimental Procedures. Acetic acid and ammonium acetate were ACS grade and diluted with deionized water. All solvents used for LC-MS or GC-MS were HPLC grade. BSTFA (N,Obis(trimethylsilyl)trifluoroacetamide; catalog no. 33155-U) was purchased from Supelco Analytical. LC-MS analysis was performed using a ThermoFinigan LCQ Advantage mass spectrometer equipped with a Surveyor Plus autosampler, MS quaternary pump, and Betasil C18 reverse-phase HPLC column (100 × 2.1 mm) with a guard column of equivalent phase. GC-MS analysis was performed with a ThermoFinigan Polaris GCQ mass spectrometer and Trace GC equipped with an Agilent DB-5MS column (30 m × 0.25 mm). Plant Material. Swainsona canescens F. Muell. and S. maccullochiana F. Muell seed lots were purchased from B & T World Seeds (Paguignan, 34210 Aigues-Vives, France). Locoweed seeds were collected from populations of Astragalus mollissimus Torr. var. earleii (Fort Davis, TX, USA, N 30°33′30.2″, W 103°51′40.5″), A. lentiginosus Douglas var. araneosus (Wahwah Valley, UT, USA, N 38°23′45.6″, W 113°17′28.66″), and Oxytropis sericea Nutt. (Park Valley, UT, USA, N 41° 54′ 15.4″, W 113° 20′ 54.9″).30 Voucher specimens of S. canescens (NSW 886571 and NSW 886572) were collected from Madoonia Downs Station, Coolgardie, Western Australia (S 31°31′06″, E 121°46′26″ and S 31°34′59″ E 122°00′11″). Plants of Swainsona canescens (n = 6) derived from the above-mentioned seeds were grown in the greenhouse with a 16 h photoperiod and day/night temperatures of 25 °C/20 °C. Leaves from the plant were harvested and frozen at −80 °C. Subsequently, the harvested leaves were freeze-dried and ground. Swainsonine (1) and DNA were extracted from this plant material for further analyses. Fungal Cultures. Scarified seeds from all taxa, as well as leaf and stem material from S. canescens, were surface sterilized in a mixture of 70% ethanol, 30% bleach, and 0.01% Triton X-100 (Promega, Madison, WI, USA) solution for 5 min, followed by a second step of 30% bleach and 0.01% Triton X-100 for 5 min, and a third step wherein the plant material was rinsed two times in sterile water. Seeds, leaves, and stems were placed on potato dextrose agar (PDA) (Becton, Dickinson and Company, Sparks, MD, USA) in polystyrene plates (Falcon) and placed in the dark at 24 °C. Fungal cultures were subcultured, with a single pinpoint mass of mycelium being placed onto the center of a preweighed sterile filter paper that was placed on the surface of a PDA plate. Cultures were grown for 41 days under conditions described above, and measurements recorded. After 41 days the culture plus filter paper and the agar were removed separately and freeze-dried. The mass of the fungal culture was calculated by subtracting the mass of the filter paper alone from the mass of the culture plus filter paper. Swainsonine amounts were determined for the fungal culture plus filter paper and agar to determine the amount produced by the endophyte. Other subcultures of the endophyte from S. canescens were grown on water agar and PDA plates at room temperature with ambient light
endophyte detection species Swainsona canescens
Swainsona maccullochiana
a
sample plant 1 plant 2 plant 3 plant 4 plant 5 plant 6 voucher NSW 886571 voucher NSW 886572 plant 1 plant 2 plant 3 plant 4
swainsonine (mg/g)
PCR culture
pg/ng
n.d.a 1.7 2.0 2.5 3.2 3.7 2.5
+ + + + + + +
− + + + + + +
