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.

Fungicide treatment and clipping of Oxytropis sericea does not disrupt swainsonine concentrations.

Swainsonine, an indolizidine alkaloid, 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 is produced by associated endophytic fungi. Prolonged consumption of swainsonine-containing plants by livestock causes a condition characterized by weight loss, depression, altered behavior, decreased libido, infertility, and death. In contrast, Astragalus and Oxytropis that do not contain swainsonine may present a valuable food source for grazing livestock in regions where palatable forage is scarce. This study tested the hypothesis that swainsonine concentrations may be reduced by fungicide treatment or by clipping, thus reducing plant toxicity. Additionally we hypothesized that clipping plants may provide a mechanism for horizontal transmission of the endophyte. To this end, four different fungicides were applied to render the endophyte non-viable, and plant vegetative tissues were periodically clipped. Treatment of Oxytropis sericea with any of four different fungicides did not alter swainsonine concentrations in plants at any of three harvest times. Additionally, we found that individual or multiple clippings had no effect on swainsonine concentrations; plants that contained swainsonine maintained concentrations, and plants low or absent in swainsonine also remained as such at each harvest. These results suggest that there is no evidence of horizontal transmission of the endophyte among individual plants due to clipping.

Development of a PCR-Based Method for Detection of Delphinium Species in Poisoned Cattle.

Toxic plants such as Delphinium spp. (i.e., larkspur) are a significant cause of livestock losses worldwide. Correctly determining the causative agent responsible for the death of an animal, whether by disease, poisonous plant, or other means, is critical in developing strategies to prevent future losses. The objective of this study was to develop an alternative diagnostic tool to microscopy and analytical chemistry to determine whether a particular poisonous plant was ingested. Polymerase chain reaction (PCR) is a tool that may allow detection of the genetic material from a specific plant within a complex matrix such as rumen contents. A pair of oligonucleotide primers specific to Delphinium spp. (i.e., larkspur) was developed; using these primers, a PCR product was detected in samples from an in vivo, in vitro, and in vivo/in vitro coupled digestion of Delphinium occidentale. Lastly, larkspur was detected in a matrix of ruminal material where the amount of larkspur was far less than what one would expect to find in the rumen contents of a poisoned animal. The PCR-based technique holds promise to diagnose larkspur and perhaps other toxic plant caused losses.

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.

The evolution of chloroplast genome structure in ferns.

The plastid genome (plastome) is a rich source of phylogenetic and other comparative data in plants. Most land plants possess a plastome of similar structure. However, in a major group of plants, the ferns, a unique plastome structure has evolved. The gene order in ferns has been explained by a series of genomic inversions relative to the plastome organization of seed plants. Here, we examine for the first time the structure of the plastome across fern phylogeny. We used a PCR-based strategy to map and partially sequence plastomes. We found that a pair of partially overlapping inversions in the region of the inverted repeat occurred in the common ancestor of most ferns. However, the ancestral (seed plant) structure is still found in early diverging branches leading to the osmundoid and filmy fern lineages. We found that a second pair of overlapping inversions occurred on a branch leading to the core leptosporangiates. We also found that the unique placement of the gene matK in ferns (lacking a flanking intron) is not a result of a large-scale inversion, as previously thought. This is because the intron loss maps to an earlier point on the phylogeny than the nearby inversion. We speculate on why inversions may occur in pairs and what this may mean for the dynamics of plastome evolution.

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.

Functional gene losses occur with minimal size reduction in the plastid genome of the parasitic liverwort Aneura mirabilis.

Aneura mirabilis is a parasitic liverwort that exploits an existing mycorrhizal association between a basidiomycete and a host tree. This unusual liverwort is the only known parasitic seedless land plant with a completely nonphotosynthetic life history. The complete plastid genome of A. mirabilis was sequenced to examine the effect of its nonphotosynthetic life history on plastid genome content. Using a partial genomic fosmid library approach, the genome was sequenced and shown to be 108,007 bp with a structure typical of green plant plastids. Comparisons were made with the plastid genome of Marchantia polymorpha, the only other liverwort plastid sequence available. All ndh genes are either absent or pseudogenes. Five of 15 psb genes are pseudogenes, as are 2 of 6 psa genes and 2 of 6 pet genes. Pseudogenes of cysA, cysT, ccsA, and ycf3 were also detected. The remaining complement of genes present in M. polymorpha is present in the plastid of A. mirabilis with intact open reading frames. All pseudogenes and gene losses co-occur with losses detected in the plastid of the parasitic angiosperm Epifagus virginiana, though the latter has functional gene losses not found in A. mirabilis. The plastid genome sequence of A. mirabilis represents only the second liverwort, and first mycoheterotroph, to have its plastid genome sequenced. We observed a pattern of genome evolution congruent with functional gene losses in parasitic angiosperms but suggest that its plastid genome represents a genome in the early stages of decay following the relaxation of selection pressures.