Isolation And Identification Of Hemerocallin-vroducing Fungal Endophyte And Its Pathological Observation In Poisoned Mice

Hemerocallis fulva L. is widely distributed in the west of China. Consumption of theroots of some Hemerocallis species causes poisoning to animals due to Hemerocallis toxicosis.The chief symptoms are blindness, neurological disease and even death, and this causessignificant economic losses. The toxicity of Hemerocallis has been traditionally attributed tothe production of an anthraquinones, hemerocallin. The mechanism underlying the toxicosis isunknown. The study collected the roots of Hemerocallis samples from Shan′xi provinces toisolate and identify the endophytic fungi. One strain of Hemerocallin-producing endophyticfungus was obtained. The toxicity of this endophytic fungus was compared with the roots ofHemerocallis toxicity using the mice as a model to observe the histopathological lesions. Thisresearch will lay the groundwork for the mechanism of hemerocallin toxicosis.1. Four strains of endophytic fungi were isolated and purified from the roots ofHemerocallis. The four isolates were detected by thin-layer chromatography and UVspectrophotometry, and then a strain of endophytic fungus produced the hemerocallin wasobtained, and named XC-1A. Morphological investigation showed that the colony of isolateXC-1A was white, round, central raised, and has aerial hyphae. ITS sequence analysis andbuilding the phylogenetic tree by Neighbor-Joining method results showed that XC-1A wasZalerion varium.2. The roots of Hemerocallis samples and the XC-1A mycelia were extracted throughheat recirculate with chloroform. UV spectrophotometry and calibration curve analysisshowed that the hemerocallin yield of the Hemerocallis roots and the XC-1A mycelia is5.54μg/g and359.88μg/g respectively.3. Mice were randomly assigned into three groups, and16mice per group. The threegroups were fed diets containing complete mice feed and the Hemerocallis roots, completemice feed and mycelium or complete mice feed at a dose of4~6g/20g body weight once daily.Feeding hemercollin LD50/15everyday, approximately0.063mg. According to thehemerocallin yield of the Hemerocallis roots and the XC-1A mycelia, mice consumedhemerocallis roots and mycelia is0.5g and0.1751g respectively。Results showed thatclinical symptoms was found obviously in mice which took whether the Hemerocallis roots or the XC-1A mycelia. The control group is normal.4. The poisoning mice exhibited spiritual depression, behabioral depression, prostrate anddifficulty eating or drinking status. Pathological examination showed that heart, liver, spleen,and kidney red-brown enlargement, cerebrum brittleness, bladder expansion and full oforange urine. The HE stained results of visceral organs and cerebrum showed: renal cells,hepatic cell swelling, rupture, alveolar wall capillary expansion and full of red blood cells,spleen lymphocytes proliferate, myocardial fibers denaturation, interstitial small blood vesselsexpansion, hyperaemia, brain tissue structure porosity, neuronal swelling.