Multiomics Reveals Mechanisms of Alternaria oxytropis Inhibiting Pathogenic Fungi in Oxytropis ochrocephala.

Endophytic fungi can benefit the host plant and increase the plant resistance. Now, there is no in-depth study of how Alternaria oxytropis (A. oxytropis) is enhancing the ability of inhibiting pathogenic fungi in Oxytropis ochrocephala (O. ochrocephala). In this study, the fungal community and metabolites associated with endophyte-infected (EI) and endophyte-free (EF) O. ochrocephala were compared by multiomics. The fungal community indicated that there was more A. oxytropis, less phylum Ascomycota, and less genera Leptosphaeria, Colletotrichum, and Comoclathris in the EI group. As metabolic biomarkers, the levels of swainsonine and apigenin-7-O-glucoside-4-O-rutinoside were significantly increased in the EI group. Through in vitro validation experiments, swainsonine and apigenin-7-O-glucoside-4-O-rutinoside can dramatically suppress the growth of pathogenic fungi Leptosphaeria sclerotioides and Colletotrichum americae-borealis by increasing the level of oxidative stress. This work suggested that O. ochrocephala containing A. oxytropis could increase the resistance to fungal diseases by markedly enhancing the content of metabolites inhibiting pathogenic fungi.

Assembly of high-quality genomes of the locoweed Oxytropis ochrocephala and its endophyte Alternaria oxytropis provides new evidence for their symbiotic relationship and swainsonine biosynthesis.

Locoweeds are perennial forbs poisonous to livestock and cause extreme losses to animal husbandry. Locoweed toxicity is attributed to the symbiotic endophytes in Alternaria sect. Undifilum, which produce a mycotoxin swainsonine (SW). We performed a de novo whole genome sequencing of the most common locoweed in China, Oxytropis ochrocephala (2n = 16), and assembled a high-quality, chromosome-level reference genome. Its genome size is 958.83 Mb with 930.94 Mb (97.09%) anchored and oriented onto eight chromosomes, and 31,700 protein-coding genes were annotated. Phylogenetic and collinearity analysis showed it is closely related to Medicago truncatula with a pair of large interchromosomal rearrangements, and both species underwent a whole-genome duplication event. We also derived the genome of A. oxytropis at 74.48 Mb with a contig N50 of 8.87 Mb and 10,657 protein-coding genes, and refined the genes of SW biosynthesis. Multiple Alternaria species containing the swnK gene were grouped into a single clade, but in other genera, swnK's homologues are diverse. Resequencing of 41 A. oxytropis strains revealed one SNP in the SWN cluster causing changes in SW concentration. Comparing the transcriptomes of symbiotic and nonsymbiotic interactions identified differentially expressed genes (DEGs) linked to defence and secondary metabolism in the host. Within the endophyte DEGs were linked to cell wall degradation, fatty acids and nitrogen metabolism. Symbiosis induced the upregulation of most of the SW biosynthetic genes. These two genomes and relevant sequencing data should provide valuable genetic resources for the study of the evolution, interaction, and SW biosynthesis in the symbiont.

Flavonoids as key bioactive components of Oxytropis falcata bunge, a traditional anti-inflammatory and analgesic Tibetan medicine.

The traditional Tibetan medicine Oxytropis falcata Bunge, in the Leguminosae family, is widely used in the west area owing to its significant anti-inflammatory and analgesic activities. O. falcata is rich in flavonoids, which are the main secondary metabolites and key bioactive components of this plant. Up to now, 91 flavonoids have been isolated from O. falcata, including isoflavone, flavone, flavonone, flavonol, homoisoflavonoid, chalcone, dihydrochalcone, chalcone dimers, and pterocarpans. The flavonoids in O. falcata have good anti-inflammatory and analgesic activities, which are comparable to those of a positive drug control (indomethacin). Furthermore, these flavonoids exhibit antibacterial, antioxidant, antitumour, anti-cardiovascular disease, and haemostatic activities. However, to date, O. falcata has not been reviewed comprehensively. Herein, the main secondary metabolites, biosynthetic pathways, and bioactivities of O. falcata are discussed.

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.

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.

Influence of phenological stage on swainsonine and endophyte concentrations in Oxytropis sericea.

Locoweeds are defined as Astragalus and Oxytropis species that cause intoxication due to the alkaloid swainsonine. Swainsonine concentrations in Oxytropis sericea were influenced by location, plant part, and the developmental stage of the plant. Concentrations followed similar trends at each location, generally increasing over the growing season in above-ground parts until the plant reaches maturity with no change in concentration in the crowns. At the onset of senescence, swainsonine decreased in floral parts to less than half of the peak concentration. Similar to swainsonine concentrations, endophyte amounts were influenced by location, plant part, and the developmental stage of the plant. Likewise, endophyte amounts generally increased over the growing season in above ground parts and remained static in the crowns at all four locations. Swainsonine in Oxytropis sericea was positively associated with the endophyte Undifilum, which is responsible for swainsonine biosynthesis.

Effects of defoliation and shading on the physiological cost of reproduction in silky locoweed Oxytropis sericea.

BACKGROUND: The production of flowers, fruits and seeds demands considerable energy and nutrients, which can limit the allocation of these resources to other plant functions and, thereby, influence survival and future reproduction. The magnitude of the physiological costs of reproduction depends on both the factors limiting seed production (pollen, ovules or resources) and the capacity of plants to compensate for high resource demand. METHODS: To assess the magnitude and consequences of reproductive costs, we used shading and defoliation to reduce photosynthate production by fully pollinated plants of a perennial legume, Oxytropis sericea (Fabaceae), and examined the resulting impact on photosynthate allocation, and nectar, fruit and seed production. KEY RESULTS: Although these leaf manipulations reduced photosynthesis and nectar production, they did not alter photosynthate allocation, as revealed by (13)C tracing, or fruit or seed production. That photosynthate allocation to reproductive organs increased >190 % and taproot mass declined by 29 % between flowering and fruiting indicates that reproduction was physiologically costly. CONCLUSIONS: The insensitivity of fruit and seed production to leaf manipulation is consistent with either compensatory mobilization of stored resources or ovule limitation. Seed production differed considerably between the two years of the study in association with contrasting precipitation prior to flowering, perhaps reflecting contrasting limits on reproductive performance.

An integrated electrocoagulation-phytoremediation process for the treatment of mixed industrial wastewater.

The elimination of organic contaminants in highly complex wastewater was tested using a combination of the techniques: electrocoagulation with aluminum electrodes and phytoremediation with Myriophyllum aquaticum. Under optimal operating conditions at a pH of 8 and a current density of 45.45 A m(-2), the electrochemical method produces partial elimination of contaminants, which was improved using phytoremediation as a polishing technique. The combined treatment reduced chemical oxygen demand (COD) by 91%, color by 97% and turbidity by 98%. Initial and final values of contaminants in wastewaters were monitored using UV-vis spectrometry and cyclic voltammetry. Finally, the morphology and the elemental composition of the biomass were characterized with using scanning electron microscopy (SEM) and energy dispersion spectroscopy (EDS). The presence of Al in the roots of plants in the system indicates that the aluminum present in the test solution could be absorbed.

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.

Relationship between the endophyte Embellisia spp. and the toxic alkaloid swainsonine in major locoweed species (Astragalus and Oxytropis).

Locoweeds (Astragalus and Oxytropis spp. that contain the toxic alkaloid swainsonine) cause widespread poisoning of livestock on western rangelands. There are 354 species of Astragalus and 22 species of Oxytropis in the US and Canada. Recently, a fungal endophyte, Embellisia spp., was isolated from Astragalus and Oxytropis spp. and shown to produce swainsonine. We conducted a survey of the major locoweeds from areas where locoweed poisoning has occurred to verify the presence of the endophyte and to relate endophyte infection with swainsonine concentrations. Species found to contain the fungal endophyte and produce substantial amounts of swainsonine were A. wootoni, A. pubentissimus, A. mollissimus, A. lentiginosus, and O. sericea. Astragalus species generally had higher concentrations of swainsonine than Oxytropis. Swainsonine was not detected in A. alpinus, A. cibarius, A. coltonii, A. filipes, or O. campestris. The endophyte could not be cultured from A. mollissimus var. thompsonii or A. amphioxys, but was detected by polymerase chain reaction, and only 30% of these samples contained trace levels of swainsonine. Further research is necessary to determine if the endophyte is able to colonize these and other species of Astragalus and Oxytropis and determine environmental influences on its growth and synthesis of swainsonine.

Seasonal fluctuations of selenium and sulfur accumulation in selenium hyperaccumulators and related nonaccumulators.

Some plants hyperaccumulate selenium (Se) up to 1% of dry weight. This study was performed to obtain insight into whole-plant Se fluxes in hyperaccumulators. Selenium hyperaccumulators Astragalus bisulcatus and Stanleya pinnata were monitored over two growing seasons for seasonal fluctuations in concentrations of Se and the chemically similar element sulfur (S). The related nonhyperaccumulators Astragalus sericoleucus, Oxytropis sericea and Thlaspi montanum were included for comparison. In both hyperaccumulators leaf Se decreased from April to October, coinciding with Se hyperaccumulation in flowers and seeds. Root Se levels were lowest in summer. Selenium concentration decreased with leaf age in both hyperaccumulators. Leaf S levels peaked in summer in all plant species, as did Se levels in nonhyperaccumulators. Selenium and S levels tended to be negatively correlated in hyperaccumulators, and positively correlated in nonhyperaccumulators. These results suggest a specific flow of Se in hyperaccumulator plants over the growing season, from root to young leaves in spring, followed by remobilization from aging leaves to reproductive tissues in summer, and back to roots in the autumn.

Effects of locoweed on serum swainsonine and selected serum constituents in sheep during acute and subacute oral/intraruminal exposure.

A study was conducted to evaluate the effects of acute and subacute locoweed exposure on serum swainsonine concentrations and selected serum constituents in sheep. Thirteen mixed-breed wethers (BW = 47.5 +/- 9.3 kg) were assigned randomly to 0.2, 0.4, or 0.8 mg of swainsonine x kg BW(-1) x d(-1) treatments. During acute (24 h) and subacute (19 d) exposure, serum swainsonine was detected in all treatments and was greatest (P < 0.03) in the 0.8 mg treatment. Serum alkaline phosphate (ALK-P) activity was increased (P < 0.01) for the 0.8 mg treatment compared with baseline (0 h) by 7 h and continued to increase throughout the initial 22 h following acute exposure to locoweed. A linear increase (P < 0.01) in serum ALK-P activity was noted, with the rate being 3.00 +/- 0.56 U x L(-1) x h(-1). Serum ALK-P activity was increased (P < 0.05) across treatments on d 7 over d -19, -12, 0, 1, 21, and 26; on d 14 over d -19, -12, 0, and 26; and on d 19 over d -19, -12, 0, 1, 21, and 26. By d 20, approximately 48 h after last exposure to swainsonine, serum ALK-P activities were no longer different (P = 0.13) than baseline (d -19, -12, and 0), and by d 26 values had generally returned to baseline. No linear (P = 0.98), quadratic (P = 0.63), or cubic effects of swainsonine with time from exposure were noted for serum aspartate aminotransferase. Similar to serum ALK-P activities, serum aspartate aminotransferase activities were increased (P < 0.05) across treatment levels on d 7, 14, 19, 20, 21, and 26 over those on d -19, -12, 0, and 1. Total serum Fe was decreased (P < 0.05) within the initial 22 h following the swainsonine exposure. On d 21 (48 h after swainsonine feeding ended), serum Fe increased to 472 mg/L. Concentrations of ceruloplasmin were lower (P < 0.10) on d 14 and 19 following exposure to locoweed. Recovery of ceruloplasmin levels coincided with similar changes in serum Fe. There was a linear (slope = 0.33 mg x dL(-1) x d(-1); P < 0.01) effect with time of exposure to locoweed (i.e., swainsonine) on serum triglyceride concentrations. Rapid changes in serum ALK-P and Fe concentrations without parallel changes in other damage markers indicate that acute exposure to swainsonine induces metabolic changes that may impair animal production and health before events of cytotoxicity thought to induce clinical manifestation of locoism.