Diversity And Detection Methods Of Fungal Endophytes From Locoweed In China

Locoweeds (Astragalus, Oxytropis and Sphaerophysa) are perennial herb flowering poisonous plantsthat induce chronic neurological disease due to the toxic alkaloid swainsonine (SW).“Locoweeds” hasbeen the primary poisonous weeds in the western natural grassland owing to the kinds of factors, such asnatural, human, shortages of fund and lagging management, and it caused the direct economic losses ofmore than billion RMB every year. The latest studies have demonstrated that endophytic fungi play theleading role in the synthetic process of locoweed toxin swainsonine, they are primary source ofswainsonine in locoweed plants. After nearly20years of studies show that endophytic fungi not onlyparticipate in swainsonine biosynthesis, but also promote the host plants growth and improve plantsresistance. This study isolated and identificated the endophytic fungi from the serious disserve locoweedsampled from Inner Mongolia and Ningxia region, and analyzed the locoweed endophytic fungi diversityuesing the biodiversity indexs. Distribution characteristics of locoweed endophytic fungi was studied bytissue section dyeing technology, and quantitative detected the concentration of Undifilum oxytropis inlocoweed tissues through the establishment of real-time fluorescent quantitative PCR. The study wouldprovide an important theoretical and technical basis for the future research of locoweed endophytic fungiand relationship between them, locoweed comprehensive prevention and control technology. This researchhas achieved following results:1. Isolation and identification of endophytic fungi from locoweedThe best isolation conditions of different tissues was determined using general tissue mass separationprocess and isolated endophytic fungi from different tissues of three plants sampled from five ecologicalcommunity under this best isolation conditions.1209strains of endophytic fungi was isolated from1819tissues, the isolation rate was66.5%. All of isolation rate were far greater than50.0%(the minimum was65.0%) but for the isolation rate of four tissues from S. salsula sampled from Bayan haot town in InnerMongolia were less than50.0%(43.7%). In all the locoweed organization, endophytic fungi colonization inthe seeds and leaves more easily.1209strains of endophytic fungi were identified for57species andbelonged to5classes,9orders (1order was uncertained),18families (4families were uncertained) and30genera by the methods of morphology combined with molecular biology.2. Diversity of locoweed endophytic fungiDistribution of endophytic fungi were more rich in leaves and seeds of S. salsula and all O. glabra DC.A. variabilis salmped from Jilantai were mainly distributed in the stems and seeds, but A. variabilis salmped from AoLun Prague were mainly distributed in the stems and leaves. The dominance species weredifferent in different plant tissues, Undifilum oxytropis, Alternaria sp. and Fusarium sp. was dominancespecies of some plant parts and even the whole plant. There was the most abundant endophytic fungidiversity in S. salsula, the diversity of endophytic fungi in A. variabilis was higher than whin the O. glabraDC (P<0.05), the diversity in O. glabra DC salmpled from BayanHot was far below the Yinchuan site(P<0.01), and the diversity of O. glabra DC was similar (P>0.05). Endophytic fungi flora distributionwas not similar in three locoweeds (P>0.05). The fungi distribution shared a high similarity from O.glabra DC between two sampling sites (P<0.01), and the Endophytic fungi isolated from A. variabilis wasalso highly similarity between two sampling sites (P<0.01), but the endophytic fungi distribution was notsimilar between the O. glabra DC and A. variabilis (P>0.05).3. Colonization situation and distribution of endophytic fungiInfection rate of endophytic fungi in all tissues from O. glabra DC and A. variabilis were above50%，this infection rate was higher than tissue mass isolation rate and the highest rate was up to100%. Theinfection rate of endophytic fungi in different tissues of O. glabra DC was seed>leaf>stem>petiole, and A.variabilis was seed>lstem>leaf, the detection rate and separation rate were similar. There was a tissueselectivity when endophytic fungi infecting the host plants, and the seeds of locoweed was main infectedparts. Endophytic fungi shared a certain extent selectivity to the host plants cell type when theycolonizating in plants. The palisade tissue and parenchyma in seed, surface cells layer that lose to the holein leaf and parenchymatous cell layer that around the edges of the vascular bundles in the stem medullalongitudinal axis was the best choice parts for endophytic fungi colonization, respectively.4. Establishment of RT-qPCR method detecting endophytic fungi in locoweedThe RT-qPCR method was successfully established to detect the endophytic fungi in locoweed. Themethod showed the high sensitivity, specificity strong, repeatability good and the detection limit was0.029pg/ng total DNA for the detection of Undifilum oxytropis in all tissues of O. glabra DC and A. variabilis.The testing found that the contents of endophytic fungi was seed>stem>leaf in O. glabra D, the contents inall tissues from Yinchuan site was higher than the same tissue from BayanHot site. The contents ofendophytic fungi was the highest in seeds and the lowest in roots of A variabilis, but the contents in alltissues from AoLun Prague site was higher than the same tissue from Jilantai site.