The filamentous fungus Fusarium graminearum possesses an RNA-interference (RNAi) pathway that acts as a defence response against virus infections and exogenous double-stranded (ds) RNA. Fusarium graminearum virus 1 (FgV1), which infects F. graminearum, confers hypovirulence-associated traits such as reduced mycelial growth, increased pigmentation and reduced pathogenicity. In this study, we found that FgV1 can suppress RNA silencing by interfering with the induction of FgDICER2 and FgAGO1, which are involved in RNAi antiviral defence and the hairpin RNA/RNAi pathway in F. graminearum. In an FgAGO1or FgDICER2-promoter/ GFP-reporter expression assay the green fluorescent protein (GFP) transcript levels were reduced in FgV1-infected transformed mutant strains. By comparing transcription levels of FgDICER2 and FgAGO1 in fungal transformed mutants expressing each open reading frame (ORF) of FgV1 with or without a hairpin RNA construct, we determined that reduction of FgDICER2 and FgAGO1 transcript levels requires only the FgV1 ORF2-encoded protein (pORF2). Moreover, we confirmed that the pORF2 binds to the upstream region of FgDICERs and FgAGOs in vitro. These combined results indicate that the pORF2 of FgV1 counteracts the RNAi defence response of F. graminearum by interfering with the induction of FgDICER2 and FgAGO1 in a promoter-dependent manner.
In 2020, severely infected fruit of pecan, Carya illinoiensis, showing distinct anthracnose symptoms were observed from pecan orchards in Uiseong (36°21'31.5"N 128°27'15.9"E) and Miryang (35°22'54.9"N 128°48'06.5"E) in South Korea. Visible symptoms occurred on fruit of the tree between June and July, which included dark, depressed and covered with irregularly shaped lesions. As the disease progressed, the lesions expanded and merged over time, leading to abscission of the fruit, which resulted in severe yield loss of pecan fruit. Of pecan varieties including Caddo, Giles and Peruque that were cultivated in each pecan orchard, Caddo appeared to be most susceptible to the disease. Estimated losses were approximately 30% and 70% for the Uiseong and Miryang pecan orchard, respectively. For pathogen isolation, ten symptomatic fruits of pecan were randomly collected and brought to the laboratory. The fruits were surface disinfested for 30 s with 70% ethanol and 1% sodium hypochlorite. These were then rinsed with sterile distilled water twice, placed in a humid chamber, and incubated at 25 ± 1°C with a photoperiod of 12 h. Acervuli filled with salmon-colored conidial masses were produced abundantly on the fruit a day after the incubation. Conidia were single celled, hyaline, cylindrical having rounded ends, smooth walls, guttulate, 15.5 to 17.7 µm long, and 3.4 to 4.8 µm wide (n = 20). Monoconidial isolates were made on 2% malt extract agar and incubated at 25°Ϲ for two weeks in the dark condition. Of those that were successfully retained, two representative isolates from each orchard were deposited in the culture collection (CDH) of the National Institute of Forest Science, Korea (Accession No. CDH2020-17–18). To ensure the identity of the pathogen, molecular identification was made based on three gene regions, the internal transcribed spacer (ITS) region of rDNA, beta-tubulin (TUB2) gene and a partial sequence of the actin (ACT), which were amplified with ITS1F/ITS4, T1/Bt2b and ACT-512F/ACT-783R, respectively (Weir et al. 2012). These were then submitted to GenBank with accession numbers of MW380423–24 for ITS, MW387129–30 for TUB2 and MW387127–28 for ACT. A BLAST search in GenBank revealed that the sequences showed high similarity to those of Colletotrichum siamense, which were identical to MT434615 and MT274214 for ITS and ACT, respectively, and 99.7% to MK967337 for TUB2. Phylogenetic analysis based on the maximum likelihood method further showed that the isolates recovered in this study clustered with C. siamense, confirming its identity. Pathogenicity was confirmed by inoculating living pecan trees. Healthy fruits from five trees were surface cleaned with cotton soaked in sterile water and air-dried. To inoculate the pathogen, three to five fruit per tree were wounded with a sterilized needle, and an aliquot of 10 μl of spore suspension (1.0 × 105 conidia/ml) of C. siamense (CDH2020-18) was dropped on each wound. To keep moisture, each treated fruit was enveloped by a plastic bag where the cotton soaked in sterile water had been placed. Controls were treated with sterile distilled water drops. The symptoms with dark, depressed and irregularly shaped lesions developed from all inoculated treatments six weeks after the inoculations, which were identical to those observed in the field. However, no symptom was observed on the control. Colletotrichum siamense was successfully re-isolated, fulfilling Koch’s postulates. Taken together, it was confirmed that C. siamense is the causal agent of anthracnose on pecan. In Korea, C. siamense was reported causing anthracnose on apple, persimmon and plum (Farr and Rossman 2020). However, to our knowledge, this is the first report of anthracnose on pecan caused by C. siamense in Korea. To control the disease effectively, more attention should be paid to other regions of the country where the disease caused by the pathogen might occur.
Fusarium graminearum virus 1 (FgV1) is a positive-sense ssRNA virus that confers hypovirulence in its fungal host, Fusarium graminearum . Like most mycoviruses, FgV1 exists in fungal cells, lacks an extracellular life cycle, and is therefore transmitted during sporulation or hyphal anastomosis. To understand FgV1 evolution and/or adaptation, we conducted mutation accumulation (MA) experiments by serial passage of FgV1 alone or with FgV2, 3, or 4 in F. graminearum . We expected that the effects of positive selection would be highly limited because of repeated bottleneck events. To determine whether selection on the virus was positive, negative, or neutral, we assessed both the phenotypic traits of the host fungus and the RNA sequences of FgV1. We inferred that there was positive selection on beneficial mutations in FgV1 based on the ratio of non-synonymous to synonymous substitutions ( d N /d S ), on the ratio of radical to conservation amino acid replacements ( p NR /p NC ), and by changes in the predicted protein structures. In support of this inference, we found evidence of positive selection only in the open reading frame 4 (ORF4) protein of DK21/FgV1 (MA line 1); mutations at amino acids 163A and 289H in the ORF4 of MA line 1 affected the entire structure of the protein predicted to be under positive selection. We also found, however, that deleterious mutations were a major driving force in viral evolution during serial passages. Linear relationships between changes in viral fitness and the number of mutations in each MA line demonstrated that some deleterious mutations resulted in fitness decline. Several mutations in MA line 1 were not shared with any of the other four MA lines (PH-1/FgV1, PH-1/FgV1 + 2, PH-1/FgV1 + 3, and PH-1/FgV1 + 4). This suggests that evolutionary pathways of the virus could differ with respect to hosts and also with respect to co-infecting viruses. The data also suggested that the differences among MA lines might also be explained by mutational robustness and other unidentified factors. Additional research is needed to clarify the effects of virus co-infection on the adaptation or evolution of FgV1 to its environments.
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