Mycoviruses From Phytopathogenic Fungi Of Botrytis

Botrytis spp is a widely spread group of plant pathogenic fungi, which causes gray mold disease of many crops and leads serious economic losses. This research focused on the characterization of five mycoviruses originated from B. cinerea, B. porri, B. squamosa, B. sinoallii and B.fabiopsis, of which two belong to Mitovirus, one belongs to Partitivirus, one is from an unassigned viral group and one that may represent a novel viral family. Two mycoviruses from those five viruses were determined to be associated with the hypovirulence of Botrytis spp, including Botrytis cinerea mitovirus1(BcMV1) from strain CanBc-1of B. cinerea and Botrytis porri RNA virus1(BpRV1) from strain GarlicBc-72of B. porri.The full length sequence of BcMV1is2084nt, which shows high sequence identity to OnuMV3b. BcMV1could be vertically and horizontally transmitted though conidia and by hyphal anastomosis. Infection of BcMV1dsRNA causes the serious debilitation of virulence and growth for virus-infected strains. TEM observation indicates the infection of BcMV1causes the swollen mitochondria, degeneration of cristae and presence of fibrous material in fungal mitochondria. In addition, BcMV1could be detected from purified mitochondria. Therefore, mitochondria are believed to be the target for BcMV1. The infection of mitochondria by BcMV1causes the unusual subcellular structure of mitochondria, which may lead the malfunction of mitochondria and causes the hypovirulence of B. cinerea. Moreover, BcMV1could generate defective interference (DI) RNA during the sporulation process of B. cinerea, designated as BcMV1-S. BcMV1-S could be horizontally transmitted. Although it inhibits the replication of BcMV1, but it does not affect the hypovirulence caused by BcMV1.BpRV1is determined to be another mycovirus conferring hypovirulence to Botrytis spp in this study. The BpRV1genome comprises two dsRNAs, dsRNA-1(6,215bp) and dsRNA-2(5,879bp), which share sequence identities of62and95%at the3'-and5'-terminal regions, respectively. Two open reading frames (ORFs), ORF I (dsRNA-1) and ORF â…¡ (dsRNA-2), were detected. The protein encoded by the3'-proximal coding region of ORF I shows sequence identities of19to23%with RNA-dependent RNA polymerases encoded by viruses in the families Totiviridae, Chrysoviridae, and Megabirnaviridae. However, the proteins encoded by the5'-proximal coding region of ORF I and by the entire ORF II lack sequence similarities to any reported virus proteins. Phylogenetic analysis showed that BpRV1belongs to a separate clade distinct from those of other known RNA mycoviruses. Purified virions of-35nm in diameter encompass dsRNA-1and dsRNA-2, and three structural proteins (SPs) of70,80, and85kDa, respectively. Peptide mass fingerprinting analysis revealed that the80-and85-kDa SPs are encoded by ORF I, while the70-kDa SP is encoded by ORF II. Introducing BpRV1purified virions into the virulent strain GarlicBc-38of B. porri caused derivative38T reduced mycelial growth and hypovirulence. These combined results suggest that BpRV1is a novel bipartite dsRNA virus that possibly belongs to a new virus family. Moreover, Botrytis squamosa RNA virus1(BsRV1) was isolated from strain LeekBc-10of B. squamosa. BsRV1and BpRV1shows highly sequence identity with91%-97%and97%-98%for nucleotide and protein sequence respectively, thus the two viruses is believed to be two strains of one viral species.The effects to host fungi were unknown for the rest three mycoviruses, although the virus infected strains showed abnormal cultural morphology. Partial genome sequence of Botrytis squamosa RNA virus2(BsRV2/Bc-21) and Botrytis fabiopsis RNA virus1(BfRV1/Bc-41) were obtained. The two viruses were isolated from strain GarlicBc-21of B. sqaumosa and strain BroadbeanBc-41of B. fabiopsis respectively. BsRV2/Bc-21and BfRV1/Bc-41are believed to be one viral species, as they showed high protein sequence identity (91%) with each other. Phylogenetic analysis indicated the two viruses were close related to FgV-DK21, and grouped with hypo viruses. However, the classification of this virus remains unknown. Moreover, another partitivirus, Botrytis fabiopsis partitivirus1(BfPV1), was also identified from strain BroadbeanBc-41, which is close related to SsPVS. Moreover, BfPV1also showed homology to a plant partitivirus of Vicia faba, VfPV-1. In addition, a mitovirus with close relationship to SsMV1was isolated from strain OnionBc-59of B. sinoallii, and named as Botrytis sinoallii mitovirus1(BsMV1).