Pathogen Identification,Biological Characteristics And Identification Of Partial Mycovirus In The Pathogen Of Curcuma Wenyujin Phoma Leaf Blight

Curcuma wenyujin Y.H.Chen et C.Ling,Curcuma L.,Zingiberaceae,is a herbaceous plant which has a long history of medicinal use in China.In 1997,Hainan Province started to introduce and cultivate C.wenyujin,with the expansion of planting area,continuous cultivation,imperfection of cultivation management and other factors,the occurrence of disease was becoming increasingly serious,which has a great impact on the yield and quality of the Chinese medicinal materials,and also has become a seriously restricts factor of the sustainable development of the C.wenyujin industry,while the quality of C.wenyujin had decreased due to Phoma leaf blight in recent years.This article first carried out preliminary studies on this disease,including pathogen identification,biological characteristics(Effects of different culture conditions on mycelial growth,fungicides screening in laboratory and conditions optimization for crude toxin-producing in vitro)and identificate a part of mycovirus,the main results are as follows:C.wenyujin Phoma leaf blight mainly damaged leaves,symptomatic plants initially had small yellowish-white lesions surrounded by yellow halos on the lower leaves.Later,the lesions developed on the upper leaf layer,and leaves turned light brown as the tissues desiccated.Black spots appeared on the upper side of leaf lesions under hot and humid conditions.Finally,the foliage wilted and the plant died.Isolation and purification of pathogenic fungi,pathogenicity test,morphological characteristics and multi-gene(ITS,TUB2 and LSU)phylogenetic analysis identified the pathogen of C.wenyujin Phoma leaf blight as Phoma matteucciicola.The results of biological characteristics test of Phoma matteucciicola strains WYJ01 and WYJ03 showed the optimum medium was PDA,the more suitable temperature range of mycelia growth for WYJ01 and WYJ03 were 28℃ and 30～32℃,respectively,the optimum pH of mycelia growth were 5.0,light condition was conducive to mycelia growth,the best carbon source were lactose,both.Sodium nitrate was the optimum nitrogen source of WYJ01 and WYJ03.In the test of inhibition effect of mycelia growth of Phoma matteucciicola in vitro,12 kinds of fungicides were used,the results showed that difenoconazole had stronger antibacterial effects for WY101,with EC50 at 0.0323 μg/mL.While for the strain WYJ03,flusilazole showed the highest inhibitory effect with EC50 at 0.0242 μg/mL.The results of the crude toxin production conditions of phytotoxins produced by Phoma matteucciicola showed that the crude toxin production ability varied significantly under different conditions between WYJ01 and WYJ03.The best crude toxin production conditions were Richards liquid culture medium,static culture for 6 d,both.The optimum temperature were 28℃ and 30℃ respectively.Phoma matteucciicola strain WYJ01 with abnormal colony morphology which grew slowly,and turned to dark easily with abundant chlamydospores,compared with other 5 strains of C.wenyujin leaf blight.The complete RNA were extracted from strains WYJ01 for high-throughput sequencing.The results showed that 7 different mycoviruses were detected from Phoma matteucciicola.A novel double-stranded RNA(dsRNA),named as Phoma matteucciicola partitivirusl(PmPV1),were used for further study.The complete genome of PmPV1 was sequenced by cDNA cloning.It has two dsRNA genome segments.The larger segment dsRNA-1(1664 bp)has a single open reading frame(ORF1)encoded a conserved RNA dependent RNA polymerase(RdRp)domain.The smaller segment dsRNA-2(1383 bp)contains a single ORF2 encoded an unknown protein.Virus particles were spherical diameter of about 30 nm.Phylogenetic analysis showed that PmPV1 is closely related to the subgroup Gammapartitivirus in the family Partitiviridae,but forming a distinct clade with Colletotrichum acutatum RNA virus 1 and Ustilaginoidea virens partitivirus 2.This is the first report of the full-length nucleotide sequence of a novel partitivirus,PmPV1,infecting P.matteuciicola strain WYJ01,the causal agent of leaf blight of Curcuma wenyujin.The research results above not only provide a theoretical basis for field control of C wenyujin disease,but also has great significance to the sustainable development of C wenyujin industry in Hainan Province.