Detection Of Ergot Alkaloid Production By Neotyphodium Gansuense In Vitro And Sequencing The DmaW Gene Of N.lolii Isolates AR1 And Lp19

Drunken horse grass (Achnatherum inebrians) is an intoxicating grass that grows in the native grasslands of Northwest China. Recent studies have shown the link between endophyte infection in A. inebrians and narcosis of grazing animals. Two major ergot alkaloids, ergine and ergonovine, have been detected in endophyte infected but not endophyte-free plants of A. inebrians. A new endophytic fungus, Neotyphodium gansuense Li and Nan, has been isolated and named but its ability to produce toxins in vitro is unknown. However, most of the multiple steps for ergot alkaloid biosynthesis in Claviceps species have been elucidated. Two important genes in this pathway are dmaW and cpd1 have been found and cloned. In this study, the production of mycelium and ergot alkaloids of 12 isolates of N. gansuense were examined in four liquid media after 4 to 8 weeks growth. The concentration and temporal variation of ergine and ergonovine of N. gansuense were assessed using HPLC. To investigate why some strains can produce ergot alkaloids but others can not. I sequenced the dmaW gene from the ergovaline pathway of N. lolii isolate Lp19 which produces ergovaline and of AR1 which does not produce ergovaline and compared them with a database sequences in GenBank.N. gansuense grew significantly better in M102 and PDB liquid media than in SM or M104. Optimal growth in liquid media was in M102. However, production of ergot alkaloids was significantly greater in SM and M104 than in the other two liquid media, and was optimal in M104.Mycelial production of 12 isolates was significantly different in M102 and PDB after four weeks growth. In M102, production by isolates NgSlal and NgL112 was the highest among the isolates tested (68.25 and 65.56 mg/ml, respectively) , isolates of NgL141and NgL221 (12.305 and 16.04 mg/ml, respectively) , which were the lowest producers at only 18.03%-27.57% and 18.77% - 24.47% of that of NgSlal andNgL112.The concentration of ergot alkaloids increased with time in SM and M104 liquid media. The ability to produce ergot alkaloids varied in the 12 isolates. After eight weeks growth in M104 medium, NgL151 produced the highest concentration of ergine (27.6 ppm) , and this was five times more than that of NgL141 (5.46ppm) which was the lowest producer. NgL221 produced the highest concentration of ergonovine (32.55 ppm), which was 10 times that of the lowest producer NgL141 (3.25 ppm) . In M104, the mean concentrations (of the twelve isolates) of ergine and ergonovine were 15.8 and 12.8 ppm respectively, 1.2 times more than those in SM (10.6 and 3.2 ppm respectively) .Ergine and ergonovine have been detected in SM and M104 liquid cultures of 11 of the 12 isolates of N. gansuense but not in the isolate of N. lolii or of isolate NgL161 of N. gansuense. Using this isolate, it may be possible to produce endophyte infected A. inebrians that are not toxic to livestock and it could provide good material for the study ofergot alkaloid biosynthesis in N. gansuense.All DNA sequences which have been cloned from the AR1 and Lpl9 isolates were the same except for four base pairs which may be the result of PCR errors.In conclusion, the characteristics of ergot alkaloid production in vitro, temporal variation and variation between isolates were characterised in this study and the dmaW gene of the ergot alkaloid biosynthesis pathway was sequenced and compared with database sequences. This has provided more basic information for further study of the N. gansuense- A. inebrians symbiota.