ObjectiveSwainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by multiple fungi. A key enzyme in the SW synthesis pathway is a hybrid swnk/nrps. To analyze the role of swnk in the SW biosynthesis pathway of Metarhizium anisopliae.ResultsThe concentration of SW and the swnk expression in M. anisopliae fermentation from 1st to 7th day were determined using LC-MS and RT-qPCR, respectively. M. anisopliae had the highest SW content and swnk expression on the 5th day of fermentation; Mutant strain (MT) were obtained by PEG-mediated homologous recombination (HR) which knocked out swnk in the wild-type (WT) strain. Complemented-type (CT) strain were obtained by transforming a modified PUC19 complementation vector containing the geneticin (G418) resistance gene and swnK. SW was not detected in the MT strain and reverted to its original level in the CT strain; A Psilent-1 plasmid with Benomyl (ben)-resistant that was used interfered with swnk of WT strain. The level of SW was markedly diminished in the RNAi strain. RNAi of swnk affects the formation of the cell wall in M. anisopliae.ConclusionThese results indicate that swnk plays a crucial role in the SW biosynthesis of M. anisopliae.

The indolizidine alkaloid, swainsonine (SW), is the main toxic component of locoweed, which can cause locoism in animals with characteristic neurological dysfunction. Pathological manifestations at cellular level include extensive vacuolar degeneration. Studies have shown that SW can induces autophagy, but the role and mechanism of autophagy in SW-induced vacuolar degeneration is unclear. In this study, we analyzed the role of autophagy in SW-induced cell injury in mouse hippocampal neurons cell line (HT22) using western blotting, qRT-PCR, transmission electron microscopy and immunofluorescence microscopy. The results showed that the expressions of LC3-II, ATG5, Beclin1 and p62 proteins and their mRNAs in HT22 cells were induced by SW treatment. The SW treatment increased the number of autophagosomes with enhanced fluorescence intensity of monodansylcadaverine (MDC) and LC3-II in a time-dose dependent manner. The results of lysosome staining showed that SW could increase the number of lysosomes, increase the intraluminal pH. Transmission electron microscopy results indicate that SW induced autophagosomes, and Baf A1 could effectively alleviate SW-induced vacuolar degeneration. At the molecular level, SW treatment inhibited the expression of p-PI3K, p-AKT, p-ERK, p-AMPK, p-mTOR, p-p70S6K and p-4EBP1 and promoted the expression of p53. Our results collectively suggest, PI3K/AKT/mTOR, ERK/mTOR and p53/mTOR signaling pathways are involved in the regulation of SW-induced autophagy in HT22 cells, while the AMPK/mTOR signaling pathway is not involved in this regulation. Inhibition of autophagic degradation can effectively alleviate SW-induced vacuolar degeneration.

Oxytropis plants are widely distributed in the grasslands in northern China. Some Oxytropis species have been reported to contain the mycotoxin swainsonine, an alkaloid which causes poisoning in livestock, referred to as locoism. Previous studies showed that endophytic fungi (Alternaria oxytropis) symbiotically associate with these Oxytropis species to produce swainsonine. However, the influence of variation within the Oxytropis genus on the fixation or loss of symbiosis and toxicity is poorly understood, as is the influence of environmental factors. Here we used a collection of 17 common Oxytropis species sampled in northern China to assess genetic diversity using genotyping by sequencing which was compared with the levels of the endophyte and swainsonine. Results showed that nine Oxytropis species have detectable A. oxytropis colonisation, and seven Oxytropis species contain sufficient swainsonine to be considered poisonous, whereas the rest may be non-toxic. Species variation rather than the genetic lineage was associated with the fixation or loss of endophyte and swainsonine production, which appears to have resulted from genetic drift. Genotype × Environment (G × E) effects were also found to influence endophyte and swainsonine levels amongst species of the Oxytropis genus. Our study will provide a better understanding about the evolutionary basis of A. oxytropis symbiosis and swainsonine biosynthesis in locoweeds.

Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by fungi including Metarhizium anisopliae , Slafractonia leguminicola , and Alternaria oxytropis . While the SW biosynthesis pathway of fungi and the catalytic enzyme genes that regulate synthesis are not cleanly. In this study, we used homologous recombination (HR) to knock out and interfere with the polyketide synthase gene ( pks ) of M. anisopliae to determine its effect on the SW biosynthesis pathway. The concentration of SW was measured in the fermentation broth of M. anisopliae at 1 d, 2 d, 3 d, 4 d, 5 d, 6 d or 7 d using LC-MS. The gene for the pks gene was detected by RT-qPCR. Day 5 of M. anisopliae gave the highest content of SW and the highest expression of the pks gene. To determine the role of the pks gene in the SW biosynthesis pathway of M. anisopliae , we used PEG-mediated homologous recombination (HR) to transform a wild-type strain (WT) with a Benomyl ( ben )-resistant fragment to knock out the pks gene producing a mutant-type strain (MT) and used PEG-mediated RNAi to transform a wild-type strain (WT) with a Benomyl ( ben )-resistant plasmid to interfere with the pks gene. A complemented-type (CT) strain was produced by adding a complementation vector that contains the geneticin (G418) resistance gene as a marker. The content of SW didn’t detected in MT strain, and returned to the original level in the CT strain, while the content of SW was significantly decreased in RNAi strain. We suggest that mutation and RNAi in the pks gene affect the cell wall formation of M. anisopliae , while the colony diameters, phenotypes, and growth rates did not change significantly, and no obvious changes in other cellular organelles were noted. These results indicate that the pks gene plays a crucial role in the SW biosynthesis of M. anisopliae , which provides an important theoretical basis for illuminating the SW biosynthesis and solving locoism in livestock.

Monocrotaline (MCT), a pyrrolizidine alkaloid, is the major toxin in Crotalaria , which causes cell apoptosis in humans and animals. It has been reported that the liver is a vulnerable target of MCT. However, the exact molecular mechanism of the interaction between endoplasmic reticulum (ER) stress and liver injury induced by MCT is still unclear. In this study, the cytotoxicity of MCT on primary rat hepatocytes was analyzed by a CCK-8 assay and Annexin V-FITC/PI assay. Protein expression was detected by western blotting and immunofluorescence staining. As a result, MCT significantly decreased the cell viability and mediated the apoptosis of primary rat hepatocytes. Meanwhile, MCT could also induce ER stress in hepatocytes, indicated by the expression of ER stress-related proteins, including GRP78, p-IRE1α, ATF6, p-eIF2α, ATF4, and CHOP. Pretreatment with 4-PBA, an inhibitor of ER stress, or knockdown of CHOP by siRNA could partly enhance cell viability and relieve the apoptosis. Our findings indicate that ER stress is involved in the hepatotoxicity induced by MCT, and CHOP plays an important role in this process.

Monocrotaline (MCT) belongs to the category of Pyrrolizdine Alkaloids (PAs), which is one of important hepatotoxic alkaloid in Crotalaria Lin. Apoptosis is one mechanism of toxic responses induced by MCT. However, the underlying mechanism of liver apoptosis caused by MCT through Endoplasmic reticulum (ER) stress continues to be incompletely understood. In this study, we describe the role of ER stress in MCT induced hepato-toxicity in rats. 24 male rats were randomly divided into 3 groups: normal saline group, 45 mg/kg MCT group and 90 mg/kg MCT group. After 48 h of saline/MCT administration, the livers were collected for analysis of ER stress-related proteins by Western blotting. The expression of GRP78, p-IRE1 alpha, ATF6 and caspase-12 showed a dose-dependent increase. PERK/eIF2 alpha/ATF4/CHOP pathway is one of the major ER stress pathways which is required for cell survival. Therefore, through analyzing the effects of MCT on this pathway, we found the protein levels of p-PERK, p-eIF2 alpha, ATF4 and CHOP were increase obviously. All these results indicate that MCT induces ER stress in rat liver. The PERK/eIF2 alpha/ATF4/CHOP pathway is involved in the regulation of MCT-induced ER stress in the liver of rat.

Monocrotaline (MCT) belongs to the category of Pyrrolizdine Alkaloids (PAs), which is one of important hepatotoxic alkaloid in Crotalaria Lin. Apoptosis is one mechanism of toxic responses induced by MCT. However, the underlying mechanism of liver apoptosis caused by MCT through Endoplasmic reticulum (ER) stress continues to be incompletely understood. In this study, we describe the role of ER stress in MCT induced hepatotoxicity in rats. 24 male rats were randomly divided into 3 groups: normal saline group, 45 mg/kg MCT group and 90 mg/kg MCT group. After 48 h of saline/MCT administration, the livers were collected for analysis of ER stress-related proteins by Western blotting. The expression of GRP78, p-IRE1α, ATF6 and caspase-12 showed a dose-dependent increase. PERK/eIF2α/ATF4/CHOP pathway is one of the major ER stress pathways which is required for cell survival. Therefore, through analyzing the effects of MCT on this pathway, we found the protein levels of p-PERK, p-eIF2α, ATF4 and CHOP were increase obviously. All these results indicate that MCT induces ER stress in rat liver. The PERK/eIF2α/ATF4/CHOP pathway is involved in the regulation of MCT-induced ER stress in the liver of rat.

Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by fungi including Metarhizium anisopliae , Slafractonia leguminicola , and Alternaria oxytropis . Studies of the SW biosynthesis pathway in these fungi have demonstrated the requirement for a swn K gene and the presence of a variety of other SWN cluster genes, but have not determined a precise role for the swnR gene, which encodes a NADB Rossmann-fold reductase, nor if it is necessary for the biosynthesis of SW. In this study, we used homologous recombination (HR) to knock out the swnR gene of M. anisopliae to determine its effect on the SW biosynthesis pathway. The concentration of SW was measured in the fermentation broth of M. anisopliae at 1 d, 3 d, 5 d and 7 d using a Q Exactive Mass Spectrometer. The gene for swnR was detected by RT-qPCR. To determine the role of the swnR gene in the SW biosynthesis pathway of M. anisopliae , we used PEG-mediated homologous recombination (HR) to transform a wild-type strain (WT) with a Benomyl ( ben )-resistant fragment to knock out the swnR gene producing a mutant-type strain (MT). A complemented-type (CT) strain was produced by adding a complementation vector that contains the glufosinate (herbicide) resistance ( bar ) gene as a marker. The content of SW decreased, but was not eliminated in the fermentation broth of the MT strain, and returned to the original level in the CT strain. These results indicate that the swnR gene plays a crucial role in the SW biosynthesis pathway of M. anisopliae , but suggests that another gene in the fungus may share the function of swnR.

Stellera chamaejasme L. (S. chamaejasme) is one of the primary toxic grass species (poisonous plants) distributed in the alpine meadows of Qinghai Province, China. In this study, according to the distinctive phenological characteristics of S. chamaejasme, the spectral differences between S. chamaejasme in the full-bloom stage and other pasture grasses were analyzed and the red, blue, and near-infrared bands of IKONOS image were determined as the diagnostic bands of S. chamaejasme recognition. Feature indexes related to S. chamaejasme were established using the diagnostic bands, and \(NDVI_{blue} = (\rho_{nir} − \rho_{blue})/(\rho_{nir} + \rho_{blue})\) obtained as S. chamaejasme sensitive index based on the linear regression analysis between the indexes derived from field spectra and the actual cover fraction of S. chamaejasme communities. The distribution area of S. chamaejasme was extracted by using the index \(NDVI_{blue}\) derived from IKONOS multispectral image in Qilian County of Qinghai Province, China and the verified result reached an overall accuracy of 90.71%. The study indicated that high resolution multispectral satellite images (such as IKONOS images) had significant potential in remote sensing recognition of toxic grass species.

Stellera chamaejasme is one of the main poisonous weeds distributed in alpine meadow of Qinghai Province. Rapid spreading of S. chamaejasme has done serious harm to local animal husbandry and caused continuous grassland ecosystem degradation. This paper focused on the spectral differences between S. chamaejasme and herbage, taking the typical degraded alpine meadow dominated by S. chamaejasme in Qilian County of Haibei Region as the test site and using the spectral measurements acquired in the full-blossom period of S. chamaejasme from 2012 to 2014. The results showed that the spectral behavior of flowers of S. chamaejasme differed significantly from green background that included leaves of S. chamaejasme and herbage within 350-900 nm of VIS-NIR wavebands. The biggest reflectance difference between flowers of S. chamaejasme and green background was located in the red valley, followed by the blue valley. The reflectance of S. chamaejasme community increased with the rising of coverage, the biggest reflectance difference between S. chamaejasme and herbage communities lied in the near-infrared peak, and the best separability between S. chamaejasme communities with different coverage was also at the point. The difference of first derivative spectra between flowers of S. chamaejasme and green background located in amplitude of yellow edge was remarkable, followed by amplitude of blue edge, the same as differences between S. chamaejasme and herbage communities. Linear regression analysis between coverage of S. chamaejasme and spectral feature parameters showed best result for red valley (R2 = 0.94). Finally, the red valley, the blue valley and the near-infrared peak were proposed for discriminating S. chamaejasme from herbage in the full-blossom period of S. chamaejasme, and the combination of corresponding red, blue and near-infrared bands could be used to build sensitive indices for S. chamaejasme recognition.