Isolation, Identification And Genetic Polymorphism Of Swainsonine-Producing Fungal Endophytes From Locoweeds In China

Locoweeds are toxic plants of the genera Astragalus and Oxytropis containing swainsonine. Locoism is one of the most widespread poisonous plant problems. Swainsonine is the principal agent responsible for locoism in animals and an potent inhibitor of lysosomeα-mannosidase and Golgi mannosidase II in cell. Inhibition of the mannosidases in cells of mammals leads to an accumulation of mannose-rich oligosaccharides in the lysosomes or interferes with the synthesis of complex glycoproteins in cells. Affected animals appear as behavioural abnormality, paralysis, infertility, abortion, weight loss and eventually death. Consumption of locoweed by animals results tremendous economic losses. Up to now, the toxicology, pathology of locoism have been detailed described, but it is remain a great problem to protect animals from this poisonous disease, locoism has become the most important disease on the vast rangeland. Recently, the fungal endophytes are isolated frequently from locoweeds in the US and showed to produce swainsonine in vitro. There is a strong relationship between the presence of endophytes and the swainsonine concentration of their host plants. The endophytes are believed to be responsible at least partially for the toxicity of locoweeds. However, it is not documented that swainsonine-producing fungal endophyte infecting locoweeds in China. The research about swainsonine-producing fungal endophyte will offer a new reference for controlling and using locoweed. This paper was main about the isolation and identification of fungal endophytes from locoweeds in China, determination of swainsonine-producing fungal endophytes, phylogenetic relationships based on ITS-5.8S rDNA sequences, genetic polymorphism and cultural characteristics on solid media of fungal endophytes.1. Isolation and identification of fungal endophytes from locoweedsTen species of the genera Astragalus and Oxytropis were conducted for isolation of fungal endophytes. Forty-two fungal isolates were obtained and classified three groups, such as Alternaria, Embellisia, and undetermined fungi. Eleven Embellisia fungi were isolated respectively from seven species of locoweeds that including Astragalus variabilis, Oxystropis glabra, Oxytropis sericopetala, Oxytropis glacialis, Oxytropis kansuensis, Oxytropis ochrocepala and Astragalus strictus. The Embellisia fungi were determined as fungal endophytes of these plants by scanning electron microscope. Moreover, fourteen Alternaria fungi and seventeen undetermined fungi were also harvested in this assay. The study was important for the further determination of swainsonine-producing fungal endophytes in these locoweeds.2. Determination of swainsonine-producing fungal endophytes from locoweedsThe methods of extraction and isolation about swainsonine were evaluate, the optimal method were determined and used to the treatment of plant and fungi samples. Eleven of 42 isolates were confirmed containing swainsonine by TLC, GC and GC-MS. The fungi containing swainsonine were Embellisia spp, isolated from Astragalus variabilis, Oxystropis glabra, Oxytropis sericopetala, Oxytropis glacialis, Oxytropis kansuensis, Oxytropis ochrocepala and Astragalus strictus, respectively. The swainsonine contents of the fungal mycelia were 10.83±1.31 mg/kg to 571.11±7.79 mg/kg. Subsequently, the fungal endophytes were also verified producing swainsonine in liquid fermentation in vitro. The concentrations of swainsonine in fermentation liquors of endophytes were 2～85 mg/L. The endophytes is promising to be used to producing swainsonine. It is the first document about swainsonine-producing fungal endophytes from locoweeds in China. The study offers a new possible avenue for dealing with locoism by controlling the endophytes instead of locoweed plants.3. Sequencing of ITS-5.8S rDNA and phylogenetic relationships based on ITS-5.8S rDNA sequences of swainsonine-producing fungal endophytesThe amplification and sequencing were conducted for the ITS-5.8S rDNA regions of eleven swainsonine-producing fungal endophytes. The sizes of amplified fragments were 600 to 602 bp. The minute differences, variations of one to three nucleotide, occurred among the ITS-5.8S rDNA sequences of eleven fungal endophytes. Phylogenetic analysis based on ITS-5.8S rDNA sequences showed the fungal endophytes were closely related to Embellisia oxytropis OIB9,Embellisia oxytropis OsL12,Embellisia oxytropis DAOM 237697,Embellisia sp. OY2.21. Eleven swainsonine-producing fungal endophytes were identified as Embellisia oxytropis according to the morphology, genetics of endophytes and associated documents. The taxa of the fungal endophytes were determined on the level of species. This study offers the basis for the further research of genetic polymorphism of swainsonine-producing fungal endophytes.4. Genetic polymorphisms of swainsonine-producing fungal endophytesThe analysis about genetic polymorphisms of swainsonine-producing endophytic fungi were performed by RAPD and IGS-RFLP. Twenty random primers were screened in this assay. It was found that a smaller difference occurred in the RAPD spectrum of different isolates only using primer OPA-1, OPA-18 and OPA-20. The results indicated that eleven swainsonine-produing fungal endophytes had a very close genetic relationship and could not be distinguished by the primers used in this study. IGS regions of all isolates had Dra I, Hind III, Sma I restriction sites and the number of restriction sites were same. However, the sizes of IGS sequences and restriction fragments from different fungi were various which exhibited the different locations of internal restriction sites. This means that there were differences among the 11 fungal species, which could be different subspecies of Embellisia oxytropis. IGS-RFLP was a potent method for distinguishing different isolates of swainsonine-producing fungal endophytes. This study provided the basis for the study on genetic variation of swainsonine-producing fungal endophytes from locoweeds.5. Characteristics of growth of swainsonine-producing fungal endophytes on solid mediaA preliminary study were performed about the Characteristics of growth of swainsonine-producing fungal endophytes (F5, G3) isolated from Oxytropis ochrocepala and Astragalus strictus on solid media. The optimal carbon source, nitrogen source, pH were oatmeal, peptone pH 4.4. The optimal ratio of carbon and nitrogen was 100:1, 200:1 respectively when inorganic nitrogen or organic nitrogen as nitrogen source. This study provides a basis for the study on the fermentation and the synthetic mechanism of swainsonine of endophytic fungi.