Review pubs.acs.org/JAFC
Swainsonine-Containing Plants and Their Relationship to Endophytic Fungi Daniel Cook,* Dale R. Gardner, and James A. Pfister Poisonous Plant Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 1150 East 1400 North, Logan, Utah 84341, United States ABSTRACT: Swainsonine, an indolizidine alkaloid with significant physiological activity, is an α-mannosidase and mannosidase II inhibitor that alters glycoprotein processing and causes lysosomal storage disease. Swainsonine is present in a number of plant species worldwide and causes severe toxicosis in livestock grazing these plants. Consumption of these plants by grazing animals leads to a chronic wasting disease characterized by weight loss, depression, altered behavior, decreased libido, infertility, and death. This review focuses on the three plant families and the associated taxa that contain swainsonine; the fungi that produce swainsonine, specifically the fungal endophytes associated with swainsonine-containing taxa; studies investigating the plant, endophyte, and swainsonine relationship; the influence of environmental factors on swainsonine concentrations in planta; and areas of future research. KEYWORDS: swainsonine, endophyte, locoweed, Ipomoea
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INTRODUCTION Several species among three genera in the Fabaceae (Fabales) family, Astragalus, Oxytropis, and Swainsona, are toxic to grazing livestock throughout the world.1−4 Many of the species within these genera are nontoxic and are important forages; however, several species are toxic to both livestock and wildlife. “Loco” is a Spanish word for crazy and has been used by the people of North America for over a century to describe the neurologic disease and behavior of poisoned animals, whereas “locoweed” has been used to describe the plants responsible for the poisoning. De Soto and other Spanish explorers in the southwestern United States first noted locoism in their horses. As settlers and stockmen moved west on the American frontier, locoweeds along with other poisonous plants emerged as a serious livestock-poisoning problem. Due to the significant number of livestock losses attributed to locoism throughout the west, the USDA Bureau of Plant Investigations established a research station at Hugo, CO, USA, in 1905.5 A subsequent series of feeding and grazing experiments conducted by C. D. Marsh demonstrated that Astragalus and Oxytropis species were the cause of locoism and documented many of the clinical symptoms associated with this neurologic disease.1,2,5 Animals intoxicated by Astragalus, Oxytropis, and Swainsona species show similar clinical signs and pathology.6,7 Swainsonine (Figure 1), an indolizidiine alkaloid, was first identified as
the bioactive principle of this neurologic disease in Swainsona canescens,8 a legume native to Australia. Swainsonine was later identified as the active principle in Astragalus lentiginosus in the United States.9 Locoweeds are defined as Astragalus and Oxytropis species that cause locoism and contain swainsonine, whereas Swainsona species that contain swainsonine are often referred to as “poison peas”. Swainsonine, an alkaloid with significant physiological activity, is an α-mannosidase and mannosidase II inhibitor that alters glycoprotein processing and causes lysosomal storage disease.8,10,11 Consumption of these plants by grazing animals leads to a chronic neurologic disease characterized by weight loss, depression, altered behavior, decreased libido, infertility, abortion, birth defects, and death.7,12 Swainsonine-containing plants are estimated to cause millions of dollars in livestock losses annually within the United States alone.13 Management solutions, intermittent drought, and a greater awareness by livestock producers have likely decreased the monetary losses; however, a recent cost analysis has not been performed. Swainsonine-containing Astragalus and Oxytropis species are primarily found throughout the temperate regions of the northern hemisphere and South America, whereas Swainsona species are found in Australia.5 Several species within each genus are reported to contain swainsonine (Table 1; Figure 2).14−25 Detection methods used include chromatography coupled with mass spectrometry,24 thin-layer chromatography,17 and a jack bean (Canavalia ensiformis) α-mannosidase inhibiton assay.14,15 The verification of swainsonine in suspected plant species should be done using chromatography coupled with mass spectrometry. Caution should be used about Special Issue: Poisonous Plant Symposium, Inner Mongolia Received: Revised: Accepted: Published:
Figure 1. Structure of the indolizidine alkaloid swainsonine. This article not subject to U.S. Copyright. Published 2014 by the American Chemical Society
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Table 1. Summary of the Astragalus, Oxytropis, and Swainsona Species Suspected of Causing Locoism and/or Reported To Have Swainsonine, Swainsonine Detection Methods, the Associated Undif ilum Endophyte (If Known), and Endophyte Detection Methods detection taxa
country
A. allochrous A. amphioxys A. asymmetricus A. bisulcatus A. didymocarpus A. drummondii A. emoryanus A. f lavus A. humistratus A. lentiginosus A. lonchocarpus A. missouriensis A. mollissimus A. nothoxys A. oxyphysus A. pehuenches A. praelongus A. pubentissimus A. purshii A. pycnostachyus A. strictus A. tephrodes A. thurberi A. variabilis A. wootoni O. besseyi O. campestris O. glabra O. glacialis O. kansuensis O. lambertii O. ochrocephala O. sericea O. sericopetala S. canescens S. galegifolia S. greyana
USA, Mexico USA USA USA USA USA USA USA USA USA, Mexico USA USA USA, Mexico USA, Mexico USA Argentina, Chile USA USA USA USA China USA USA China USA, Mexico USA USA, Canada China China China USA, Canada China USA, Canada China Australia Australia Australia
swainsonine detection
endophyte
enzyme LC-MS TLC TLC, enzyme TLC enzyme TLC, enzyme, MS TLC
culture
Undif ilum sp.
TLC, LC-MS enzyme enzyme TLC, enzyme, LC-MS TLC TLC TLC/enzyme LC-MS TLC GC-MS enzyme enzyme GC-MS, LC-MS TLC, LC-MS
GC-MS, LC-MS GC-MS GC-MS TLC, enzyme, GC-MS, LC-MS GC-MS TLC, enzyme, LC-MS GC-MS GC-FID, LC-MS GC-FID GC-FID
■
PCR x
Undif ilum f ulvum
x
x
Undif ilum cinerum
x
x
Undif ilum sp.
x
x
Undif ilum oxytropis
x
Undif ilum oxytropis Undif ilum sp.
x x
x x
Undif ilum Undif ilum Undif ilum Undif ilum Undif ilum Undif ilum Undif ilum Undif ilum
x x x x x x x x
x
oxytropis oxytropis oxytropis oxytropis oxytropis oxytropis oxytropis sp.
x x x
refs 14, 15 16 17 14, 15, 17 17 14, 15 17, 18 17 19 16, 17 14, 15 14, 15 14−17 20 17 21 14, 15, 17 16 19 17 22 14, 15 14, 15 22, 23 16, 17 20 20 22, 23 22 22 14, 15, 17, 24 22 14−17, 24 22 25, 37 25 25
OTHER SWAINSONINE-CONTAINING PLANTS In addition to the Fabaceae family, swainsonine has been documented in two other plant families: the Convolvulaceae (Solanales) and the Malvaceae (Malvales) (Figure 2; Table 3). Swainsonine was identified in these plant families after numerous episodes of livestock poisoning by these plants and subsequent economic impact. Many of the species within these families are nontoxic and are important forages. Clinical signs and pathology of animals poisoned by these plants are similar to those of animals poisoned by swainsonine-containing legumes.6,26−31 Two genera in the morning glory family, Ipomoea and Turbina, contain swainsonine. Seven taxa among these two genera, Ipomoea carnea, Ipomoea polpha, Ipomoea riedelii, Ipomoea sericophylla, Ipomoea sp. Q6 (aff. calobra), Ipomoea verbascoidea, and Turbina cordata, contain swainsonine (Table 2).26−30 Swainsonine concentrations also vary considerably in some of these species.30 These species are found in South America or Australia with the exception of I. carnea and I. verbascoidea. I. carnea can be found throughout the tropics
the presence of swainsonine without this verification as the other methods are preliminary and not definitive. Swainsonine concentrations in locoweeds vary among species, varieties, and populations, often ranging from not detected to >0.2%.16,24 For example, in Astragalus amphioxys mean swainsonine concentrations were 0.1%.16 Additionally, in some varieties of Oxytropis lambertii and A. mollissimus, such as O. lambertii var. articulata, O. lambertii var. lambertii, and A. mollissimus var. thompsonii, mean swainsonine concentrations were 0.01%.16,24 The purpose of this review is to highlight past and current research investigating (1) other swainsonine-containing plants besides those in the Fabaceae family; (2) fungi that produce swainsonine; (3) the plant, endophyte, and swainsonine relationship; (4) environmental factors that influence swainsonine concentrations; and (5) areas for future research. 7327
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Table 3. Important Characteristics of Plant−Fungal Symbiota Producing Swainsonine Considered in This Review Fabaceae host clade host growth form
eudicots herbaceous
host genera
Astragalus, Oxytropis, and Swainsona Pleosporales/ Pleosporaceae strictly vertical transmission (?)
fungal order/ family endophyte transmission
Convolvulaceae
Malvaceae
eudicots mostly woody vines (also shrubs, trees, herbaceous vines) Ipomoea and Turbina
eudicots herbaceous
Chaetothyriales/ Chaetothyriaceae (?) strictly vertical transmission (?)
?
Sida
?
Undifilum oxytropis (Pleosporales), an endophyte associated with O. lambertii, Oxytropis kansuensis, and Oxytropis sericea, is reported to produce swainsonine (Table 1; Figure 2).33,34 Initial studies described this endophyte as an Embellisia species.35 The Undif ilum genus (Pleosporales) is phylogenetically related to the genera Alternaria, Embellisia, and Ulocladium.34 On the basis of molecular and morphological characteristics, three unique Undif ilum species, Undifilum cinerum, Undifilum fulvum, and Undifilum oxytropis, have been found and identified among the three North American locoweed taxa A. mollissimus, A. lentiginosus, and O. sericea, respectively (Table 1).34,36 This is in contrast to the swainsonine-containing Astragalus and Oxytropis species examined to date in China, which all have the same endophyte species, U. oxytropis (Table 1).22 A swainsonine-producing Undif ilum sp. has also been identified in the Australian species S. canescens (Table 1); molecular and morphological data suggest it is a novel species as well.37 To date, the swainsoninecontaining Astragalus, Oxytropis, and Swainsona spp. evaluated for the presence of an Undif ilum spp. by PCR are associated with an Undifilum spp. (Table 1).16,22,37 Undif ilum spp. can be cultured from seeds and above-ground plant tissues; however, they have not been cultured from below-ground tissues.33 Undif ilum spp. are primarily found in the seed coat as they can be cultured from the seed coat but not the embryo.38 As a result, plants derived from the embryo where the seed coat has been removed contain little or no swainsonine.38 Using microscopy, U. oxytropis was detected growing between cells in vegetative tissues of O. sericea, providing further support that it is an endophyte.39 Recently, a swainsonine-producing endosymbiont in the ascomycete order Chaetothyriales was isolated from I. carnea (Table 1).40 This endosymbiont was cultured from seeds and
Figure 2. Photographs of swainsonine-containing plants: (A) Oxytropis lambertii; (B) Astragalus lentiginosus; (C) Astragalus mollissimus; (D) Swainsona galegifolia; (E) Ipomoea carnea; (F) Sida carpinifolia. Photographs A, B, and C were provided courtesy and copyright of Al Schneider, http://www.swcoloradowildflowers.com; photograph D was provided courtesy of Moonlight0551, http:// commons.wikimedia.org; photograph E was provided courtesy of J. M. Garg, http://commons.wikimedia.org; and photograph F was provided courtesy of Franklin Riet-Correa.
worldwide, whereas I. verbascoidea is an African species by herbarium record,32 although the report of its containing swainsonine comes from Brazil.29 Only a single species of the Malvaceae, Sida carpinifolia, contains swainsonine (Table 2).31
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SWAINSONINE-PRODUCING FUNGI For many years it was thought that swainsonine was a plantderived secondary metabolite; however, recent research suggests otherwise. A swainsonine-producing ascomycete,
Table 2. Summary of the Ipomoea, Turbina, and Sida Species Reported To Have Swainsonine, Swainsonine Detection Methods, the Associated Endosymbiont (If Known), and Endophyte Detection Methods detection taxa
country
swainsonine detection
endophyte
culture
PCR
refs
I. carnea I. polpha I. riedelii I. sericophylla I. sp. Q6 (aff. calobra) I. verbascoidea T. cordata S. carpinifolia
tropics worldwide Australia Brazil Brazil Australia Brazil and Africa Brazil Brazil
GC-MS, LC-MS GC-MS LC-MS LC-MS GC-MS LC-MS LC-MS LC-MS
Chaetothyriales sp.
x
x
26, 40 27 28 28 27 29, 32 30 31
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Other factors include, but are not limited to, different plant species, varieties, and populations as well as the respective endophyte associated with each. Secondary metabolites often differ among plant parts and as a function of plant phenology. Swainsonine is found in all plant parts with greater concentrations in above-ground parts than in below-ground parts in Astragalus spp. and O. sericea (Figure 4A).45,46 Furthermore, swainsonine concentrations vary season-
plants and detected by PCR in plants containing swainsonine. Unlike the locoweed endophyte, this endosymbiont grows as an epiphyte on the leaf surface of plants containing swainsonine (Figure 3). The epiphyte/endosymbiont was not observed on
Figure 3. Photograph of Ipomoea carnea leaf showing the Chaetothyriales fungal endosymbiont that produces swainsonine.
the surface of leaves from plants lacking swainsonine that were derived from fungicide-treated seeds. Plants derived from fungicide-treated seeds did not contain swainsonine but rather contained calystegines, nortropane alkaloids that are also glycosidase inhibitors. The calystegines are also found at similar concentrations in the control non-fungicide-treated plants.40 Swainsonine has been detected in other phylogentically disjunct groups of Basidiomycetes and/or Ascomycetes. Rhizoctonia leguminicola (Cantharellales), a basidiomycete, is a fungal pathogen of red clover (Trifolium pratense) that causes black patch disease.41 R. leguminicola produces swainsonine as well as the indolizidine alkaloid slaframine, which is responsible for the slobbers syndrome in horses.41,42 Recent data suggest that this fungus is misclassified and is actually an ascomycete (R. Creamer, personal communication). Metarhizium anisopliae (Hypocreales), an ascomycete, produces swainsonine and is an entomopathogen that attaches to the outside of an insect, grows internally, and causes death.43 Lastly, a preliminary study suggests two ascomycetes, Fusarium trinctum and a Schizophyllum sp., may also produce swainsonine.44 These fungi were isolated from Chinese swainsonine-containing Astragalus and Oxytropis species and their rhizosphere soils. More research is needed to determine if F. trinctum and the Schizophyllum sp. produce swainsonine as there was no detailed analytical work performed such as mass spectrometry coupled to liquid or gas chromatography.
Figure 4. (A) Swainsonine concentrations (%) and (B) endophyte amounts (pg/ng total DNA) in different plant parts of Oxytropis sericea from Cuchara, CO, USA, as a function of phenological stage. Mean swainsonine concentrations and endophyte amounts ± the standard error at the different phenological stages (stage 1, vegetative; stage 2, early flower; stage 3, late flower/early pod; stage 4, full pod/mature seed; stage 5, senesced) are shown. Adapted from data presented in Cook et al.47
ally or as a function of phenology.47,48 In O. sericea, swainsonine concentrations increase in above-ground tissues throughout the growing season until the plant reaches maturity (Figure 4A).47 Young newly developing tissues have the least amount of swainsonine, whereas more mature tissues have more swainsonine.47 As plants senesce, swainsonine concentrations decrease significantly.47 Likewise, the endophyte Undifilum spp. is found in all plant parts, again with greater concentrations in above-ground parts than below-ground parts with the exception
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PLANT/ENDOPHYTE RELATIONSHIP Multiple factors may influence the interaction of plant and endophyte and ultimately determine swainsonine concentrations in planta. Some considerations include, but are not limited to, different plant parts and the phenological stages of the plant. 7329
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Figure 5. Swainsonine concentrations (%): (A) field collections from two populations, Cuchara, CO, USA (C), and Park Valley, UT, USA (PV), of Oxytropis sericea; (B) common garden performed in greenhouse of two O. sericea populations; (C) common garden of fungal endophyte isolated from two O. sericea populations; (D) common garden of cross inoculation experiment (location: plant/endophyte). Figure adapted from data presented in Cook et al.49
of the root crown (Figure 4B).45,46 The root crown has endophyte amounts more similar to those of above-ground parts, and because many locoweeds are perennial plants, this may serve as a reservoir for the endophyte for the following year’s growth.45,46 In O. sericea, endophyte amounts increase in above-ground parts throughout the growing season until the plant reaches maturity (Figure 4B).47 Swainsonine and endophyte amounts are highly correlated over the growing season in O. sericea, indicating that endophyte amount plays a critical role in determining swainsonine concentration in the plant.47,48 Environment and genotype of the plant and/or endophyte or an interaction of these factors may influence swainsonine concentrations. Swainsonine concentrations differ spatially among populations of O. sericea; for example, mean concentrations were approximately 3 times greater in plants from Cuchara, CO, USA (0.40%), compared to plants from Park Valley, UT, USA (0.15%) (Figure 5A).47 The following observations suggest that concentration differences between these populations are due to the genotype of the endophyte. First, differences in swainsonine concentrations between populations persisted when plants from these two locations were grown in a common garden (Figure 5B).49 Second, the respective endophyte, U. oxytropis, from each population produced different amounts of swainsonine in vitro when grown under common culture conditions (Figure 5C).49 Third, a cross inoculation experiment, wherein the endophyte from one population was inoculated onto the embryo from the other location, demonstrated that the endophyte genotype, not the
plant genotype nor differences in endophyte amount, were responsible for differences in swainsonine concentration between the two populations (Figure 5D).49 Although no differences were observed in the internal transcribed spacer sequence between endophyte isolates from the two populations,49 other genotypic differences may be present that explain the difference in swainsonine production. Data suggest that the interaction of the plant species and endophyte genotype is a key factor in determining swainsonine concentrations in planta. First, different Astragalus and Oxytropis species had similar rank order swainsonine concentrations in field collections and greenhouse-grown plants. For example, Astragalus wootoni had the highest mean swainsonine concentration followed by similar concentrations in A. lentiginosus and A. mollissimus var. mollissimus, and the lowest concentrations were noted in O. sericea.16,50 Second, similar trends in rank order swainsonine concentrations between field collections and greenhouse-grown plants were observed from the highest to lowest, respectively, in A. mollissimus var. mollissimus, O. sericea, A. mollissimus var. bigelovii, and A. mollissimus var. matthewsii or var. thompsonii.51,52 In the latter example the trends in rank order swainsonine concentrations remained the same whether or not the plants were grown under optimum conditions, under water stress, or with a deficiency in nitrogen.51,52 It is not known if the respective endophyte from each of above species produces different amounts of swainsonine; however, results observed for swainsonine production between the U. oxytropis isolates found in the Cuchara, CO, USA, and Park Valley, UT, USA, O. sericea 7330
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populations suggest this may be the case.49 Caution should be used not to extrapolate beyond the populations of the highlighted taxa because other populations of these same taxa may have different mean swainsonine concentrations. Swainsonine concentrations differ among individuals within toxic populations of Astragalus and Oxytropis spp.16,24 Swainsonine concentrations segregate into two groups, namely, chemotype 1 plants, which have swainsonine concentrations >0.01%, and chemotype 2 plants, which have concentrations much
