Identification of Mycoviruses by dsRNA Extraction.

Mycoviruses exist in all major groups of fungi. With the continuous development of science and technology, the methods of studying viruses are constantly updated, and progressively mycoviruses have been discovered where most of these viruses are RNA viruses. Therefore, double-stranded RNA has traditionally been used as the hallmark of RNA mycovirus detection. This report describes in detail the method of mycovirus identification using extraction of dsRNA. Besides, extraction of viral dsRNA, and the assembly methods of viral genome and identification of virus type are presented.

A Mycovirus VIGS Vector Confers Hypovirulence to a Plant Pathogenic Fungus to Control Wheat FHB.

Mycovirus-mediated hypovirulence has the potential to control fungal diseases. However, the availability of hypovirulence-conferring mycoviruses for plant fungal disease control is limited as most fungal viruses are asymptomatic. In this study, the virus-induced gene silencing (VIGS) vector p26-D4 of Fusarium graminearum gemytripvirus 1 (FgGMTV1), a tripartite circular single-stranded DNA mycovirus, is successfully constructed to convert the causal fungus of cereal Fusarium head blight (FHB) into a hypovirulent strain. p26-D4, with an insert of a 75-150 bp fragment of the target reporter transgene transcript in both sense and antisense orientations, efficiently triggered gene silencing in Fusarium graminearum. Notably, the two hypovirulent strains, p26-D4-Tri101, and p26-D4-FgPP1, obtained by silencing the virulence-related genes Tri101 and FgPP1 with p26-D4, can be used as biocontrol agents to protect wheat from a fungal disease FHB and mycotoxin contamination at the field level. This study not only describes the first mycovirus-derived VIGS system but also proves that the VIGS vector can be used to establish multiple hypovirulent strains to control pathogenic fungi.

The Wheat Endophyte Epicoccum layuense J4-3 Inhibits Fusarium graminearum and Enhances Plant Growth.

Fungal endophytes are well-known for their ability to promote plant growth and hinder fungal diseases, including Fusarium head blight (FHB) caused by Fusarium graminearum. This study aimed to characterize the biocontrol efficacy of strain J4-3 isolated from the stem of symptomless wheat collected from Heilongjiang Province, China. It was identified as Epicoccum layuense using morphological characteristics and phylogenetic analysis of the rDNA internal transcribed spacer (ITS) and beta-tubulin (TUB). In a dual culture assay, strain J4-3 significantly inhibited the mycelial growth of F. graminearum strain PH-1 and other fungal pathogens. In addition, wheat coleoptile tests showed that lesion symptoms caused by F. graminearum were significantly reduced in wheat seedlings treated with hyphal fragment suspensions of strain J4-3 compared to the controls. Under field conditions, applying spore suspensions and culture filtrates of strain J4-3 with conidial suspensions of F. graminearum on wheat spikes resulted in the significant biocontrol efficacy of FHB. In addition, wheat seedlings previously treated with spore suspensions of strain J4-3 before sowing successfully resulted in FHB reduction after the application of conidial suspensions of F. graminearum at anthesis. More importantly, wheat seedlings treated with hyphal fragments and spore suspensions of strain J4-3 showed significant increases in wheat growth compared to the controls under greenhouse and field conditions. Overall, these findings suggest that E. layuense J4-3 could be a promising biocontrol agent (BCA) against F. graminearum, causing FHB and a growth-promoting fungus in wheat.

Interference of Small RNAs in Fusarium graminearum through FgGMTV1 Infection.

Small RNA (sRNA) plays a central role in RNA silencing in fungi. The genome of Fusarium graminearum gemytripvirus 1 (FgGMTV1) is comprised of three DNA segments: DNA-A, DNA-B, and DNA-C. DNA-A and DNA-B are associated with fungal growth and virulence reduction. To elucidate the role of RNA silencing during the interactions of fungi and viruses, the sRNA profiles of F. graminearum in association with FgGMTV1 were established, using an FgGMTV1-free library (S-S), a library for infection with the DNA-A and DNA-B segments (S-AB), and a library for infection with the DNA-A, DNA-B, and DNA-C segments (S-ABC). A large amount of virus-derived sRNA (vsiRNA) was detected in the S-AB and S-ABC libraries, accounting for 9.9% and 13.8% of the total sRNA, respectively, indicating that FgGMTV1 triggers host RNA silencing. The total numbers of sRNA reads differed among the three libraries, suggesting that FgGMTV1 infection interferes with host RNA silencing. In addition, the relative proportions of the different sRNA lengths were altered in the S-AB and S-ABC libraries. The genome distribution patterns of the mapping of vsiRNA to DNA-A and DNA-B in the S-AB and S-ABC libraries were also different. These results suggest the influence of DNA-C on host RNA silencing. Transcripts targeted by vsiRNAs were enriched in pathways that included flavin adenine dinucleotide binding, protein folding, and filamentous growth.

Fungal Virus, FgHV1-Encoded p20 Suppresses RNA Silencing through Single-Strand Small RNA Binding.

Fungal viruses are widespread in fungi infecting plants, insects and animals. High-throughput sequencing has rapidly led to the discovery of fungal viruses. However, the interactive exploration between fungi and viruses is relatively limited. RNA silencing is the fundamental antivirus pathway in fungi. Fusarium graminearum small RNA (sRNA) pattern was regulated by Fusarium graminearum hypovirus 1 (FgHV1) infection, indicating the activation of RNA silencing in virus defense. In this study, we focused on the function of an uncharacterized protein sized at 20 kD (p20) encoded by FgHV1. In the agro-infiltration assay, p20 was identified as a novel fungal RNA silencing suppressor. p20 can block systemic RNA silencing signals besides local RNA silencing suppression. We further elucidated the RNA silencing suppression mechanism of p20. The single-strand sRNA, instead of double-strand sRNA, can be incorporated by p20 in electrophoretic mobility shift assay. p20 binds sRNA originating from virus and non-virus sources in a non-sequence-specific manner. In addition, The F. graminearum 22 and 23-nt sRNA abundance and pathways related to RNA processing and redox regulation were regulated by p20. Our study revealed the first fungal virus-encoded RNA silencing suppressor with sRNA binding capability.

Effects of Seasonal Variation on the Alkaloids of Different Ecotypes of Epichloë Endophyte-Festuca sinensis Associations.

The Epichloë endophyte-Festuca sinensis association produces alkaloids which can protect the host plant from biotic and abiotic stresses. Alkaloid concentrations depend on the genetic predisposition of grass and endophyte, and are affected by the environment. Endophyte infected F. sinensis of six ecotypes were grown in experimental field and greenhouse for 2 years. Their aboveground plant tissues were collected each season to test for peramine, lolitrem B, and ergot concentrations. The results showed that seasonal changes affected the peramine, lolitrem B and ergot concentrations of Epichloë endophyte-F. sinensis associations; and these three different alkaloids responded differently to seasonal variation. The peramine concentration of six ecotypes of F. sinensis decreased significantly (p < 0.05) from spring to autumn. The lolitrem B concentration of F. sinensis was higher in autumn than in other seasons. Ergot concentrations of five ecotypes (41, 57, 84, 99, and 141) of F. sinensis peaked in the summer, and lowered in spring and autumn. In addition, the ecotype has insignificant effect (p > 0.05) on the peramine and lolitrem B concentrations of F. sinensis, but it has a significant impact (p < 0.05) on the ergot concentrations. We concluded that the seasonal variation and ecotypes can influence the alkaloids produced by the F. sinensis-endophyte associations, but the effects of seasonal conditions on the alkaloid concentrations are more pronounced than ecotypes.