Ustiloxins From Rice False Smut Balls And Their Biological Activities

Rice false smut is one of the main fungal diseases on rice.It was caused by the fungal pathogen Villosiclava virens Tanaka and Tanaka(anamorph:Ustilaginoidea virens Takahashi)and formed rice false smut balls(FSBs)in the rice panicles.It has been reported that there are three kinds of mycotoxins in FSBs,one of them are ustiloxins.Ustiloxins attracted much attention with their unique cyclopeptide structures with ether bonds in the core ring as well as strong phytotoxic,animal toxic and cytotoxic activities.In this study,several more-polar secondary metabolites including ustiloxins were isolated from rice FSBs.Their structures were elucidated,and biological activities were evaluated.Furthermore,the contents of ustiloxins in rice FSBs were quantitatively analyzed,and the effects of ustiloxins on the activities of several defense-related enzymes in rice suspension cultured cells and plants were also investigated.The main results are as follows.(1)Nine compounds were isolated from the water extract of rice FSBs by a series of chromatographic methods.They were identified as ustiloxins A,B.D(Uvb-10),F(Uvb-8)and G(Uvb-3),as well as naphthisoxazol A(Uvb-1),(3S)-1,2,3,4-tetrahydro-?-carboline-3-carboxylic acid(Uvb-4),1-methyl-1,2,3,4-tetrahydro-?-carboline-3-carboxylic acid(Uvb-7)and ?-methylphenylalanine(Uvb-9).Among five identified ustiloxins,ustiloxin G was a new compound.(2)The ustiloxins were evaluated for their phytotoxicity and cytotoxicity.Ustiloxins A,B and G showed strong inhibitory activities on seed radicle and plumule elongation of two rice varieties,Lijiang and Zhonghua 11.The inhibition ratios of the ustiloxins on radicle elongation were stronger than those on plumule elongation.Their inhibition ratios reached more than 90%on the radicle elongation,and more than 50%on the plumule elongation at concentrantion of 200 ?g/mL.The short and swelling radicles and plumules were observed.Ustiloxin A showed cytotoxic activity against tumor cell lines of BGC-823 and A549 with IC50 values of 2.66 and 3.12 ?M,respectively.Ustiloxin B showed cytotoxic activity against BGC-823,HCT-116,NCI-H1650 and HepG2 cells with IC50 values of 1.03,7.2,21.6 and 13.0 ?M,respectively,and ustiloxin G showed weak activity against cell lines A549 and A375 with IC50 values of 36.5 and 22.5 ?M,respectively.(3)The rice FSBs with their different maturity and distribution were quantitatively analyzed for ustiloxins A and B.For all samples,the content of ustiloxin A was higher than ustiloxin B.For the whole rice FSBs,the total yield of ustiloxins A and B in the early maturity stage(63.4 ?g)was much higher than that in the middle and late maturity stage(24.3 ?g and 31.2 ?g).For the same maturity of rice FSBs,the total yield of ustiloxin A and B in middle layer was much higher than the other part.The relative content of ustiloxin B in early maturity stage was higher than that in middle and late maturity stage.14 rice FSB samples collected from 8 provinces in different years were analyzed for the contents of ustiloxins A and B.The average weight of each rice FSBs varied greatly,and the content of ustiloxin A was higher than that of ustiloxin B.However,there was no evidence to show that ustiloxin contents in matured rice FSBs were correlated to the collection places,collection times,and average weight of each FSB.(4)The sclerotium and rice FSBs with sclerotium were quantitiative analyzed for ustiloxins A and B.For the rice FSBs with sclerotium,the size of sclerotium varied greatly,there was no significant difference of the total ustiloxins A and B content between sclerotium and mycelia,the values were varied from 2124.9 and 2076.2 ?g/g.In addition,the relative content of ustiloxin B in sclerotium was higher than mycelia.The sclerotium and rice FSBs with sclerotium were identified for the volatile composition.There were three compounds identified from sclerotium,the kinds of compounds in endosperm were the most,the relatice content of palmitic acid and oleic acid were higher than other composition in all the samples.(5)The effects of crude ustiloxin extract,ustiloxins A and B on the activities of defense-related enzymes including phenylalanine ammonia lyase(PAL),peroxidase(PPO)and polyphenol oxidase(POD)in rice suspension cultured cells and plants were studied.All the tested concentrations of ustiloxins A and B inhibited PAL activity of rice cells at 72 and 96 h,while all the three samples increased the PAL activity of rice plants,ustiloxin B can increased rice plants PAL activity for 3?4 fold than control at 24 h and 5?20?g/mL.The three samples increased PPO activity of rice cultured cells weakly,however,there was no obvious effect on rice plants PPO activity.The ustiloxin crude extract showed weakly inhibitory effects on POD activity of rice cultured cells and plants,the effects of ustiloxin A were opposite to ustiloxin B on POD activity of rice cells and plants,ustiloxin B increased the POD activity of rice cultured cells and plants significantly,for 40?g/mL,the POD activity of rice cultured cells was increased 5.33 and 4.35 fold of control at 24 h and 48 h,respectively.In this study,a new ustiloxin namely ustiloxin G was revealed from rice FSBs.Their biological activities including phytotoxic and cytotoxic activities were evaluated.The contents and distribution of ustiloxins in rice FSBs were analyzed.The effects of ustiloxins on the activities of three defense-related enzymes in rice plants and suspension cultured cells were also strudied.The results will enrich ustiloxin diviersity,and provide the basic data to clarify biosynthesis and distribution of ustiloxins in rice FSBs as well as the physiological functions in the interactions between the pathogen Villosiclava virens and rice plants.