Hypovirulence of Colletotrichum gloesporioides Associated with dsRNA Mycovirus Isolated from a Mango Orchard in Thailand
<p>Colony morphology of anthracnose causing disease, <span class="html-italic">C. gloeosporioides</span>, and mycovirus dsRNA profiles. (<b>A</b>) Morphology of <span class="html-italic">C. gloeosporioides</span> strain Ssa-44.1 following 5 days incubation at 25 °C on PDA at 5 days after incubation. (<b>B</b>) Presence of mycovirus confirmed by gel electrophoresis on 1% agarose gel. Lane M, marker (GeneRuler 1 kb DNA Ladder).</p> "> Figure 2
<p>Schematic representation of mycovirus genome strain Ssa-44.1 and phylogenetic analysis, (<b>A</b>) The genetic organization of mycovirus Ssa-44.1 is depicted in this illustration. The genome of the mycovirus Ssa-44.1 is 2939 bp long and contains two ORFs (ORF-1 and ORF-2). The brown and green rectangles represent open reading frames (ORFs) encoding a hypothetical protein and the RdRp, respectively. (<b>B</b>) The maximum likelihood method was used to create a phylogenetic tree based on the alignment of complete RdRp aa sequence together with previously reported members of the genera <span class="html-italic">Partitivirus</span>, <span class="html-italic">Chrysovirus</span>, <span class="html-italic">Totivirus,</span> and Unclassified dsRNA viruses. The percentage of trees in which the associated taxa clustered together is shown next to the branches. In the phylogenetic tree, CgRV1-Ssa-44.1 ORF 2 encodes the RdRp and is represented by a red letter; genetic distance substitutions are displayed by scale bars.</p> "> Figure 3
<p>CgRV2’s dsRNA elimination and biological comparison between virus-infected and isogenic virus-free strains. (<b>A</b>) Confirmation of the presence of double-stranded RNA between virus-infected CgRV2 and isogenic virus-free strain Ssa-44.1#18 using 1% gel electrophoresis and RT-PCR methods with primers generated from ORF 1 and 2 of CgRV2 sequence. Lane M, marker (GeneRuler 1 kb DNA Ladder). (<b>B</b>–<b>E</b>) Colony morphology (<b>B</b>,<b>D</b>) and growth area (<b>C</b>,<b>E</b>) of fungi when grown at 25 °C on PDA (<b>B</b>,<b>C</b>) and Vogel’s agar (<b>D</b>,<b>E</b>) for 5 days. The bars indicate standard deviation from five replicates and asterisks indicate that there was significant difference at <span class="html-italic">p</span> < 0.05 using one-way ANOVA analysis (<b>C</b>,<b>E</b>).</p> "> Figure 4
<p>Transmission of CgRV1-Ssa-44.1 to <span class="html-italic">C. gloeosporioides.</span> (<b>A</b>) Horizontal transmission via co-culturing of virus-infected strain Ssa-44.1 as a donor and virus-free strain Ssa-44.1#18 as a recipient on PDA for 6 days at 25 °C. The transmission of the virus from donor to recipient fungus is indicated by an arrow. Number (1, 2, and 3) indicates the position where the mycelia of fungal recipients were taken for subsequent culture. (<b>B</b>) Double-stranded RNA fractionation on 1% agarose gel. Lane M, marker (GeneRuler 1 kb DNA Ladder). Each lane represents an independent fused colony. (<b>C</b>) Colony morphology of fungi when grown at 25 °C on PDA for 5 days. (<b>D</b>) Vertical transmission of CgRV1-Ssa-44.1 via spore. Each single spore isolate was checked for the presence of dsRNA; the figure shows the presence or absence of dsRNA in each replicate.</p> "> Figure 5
<p>Virulence assay of virus-infected and virus-free <span class="html-italic">C. gloeosporioides</span> on mango. (<b>A</b>) The images illustrate representative lesions elicited by the fungal strains 7 days after mango leaf inoculation. A white circle was used to represent the symptom zones, which were enlarged at the bottom of each leaf image. (<b>B</b>) The images illustrate representative lesions elicited by the fungal strains 7 days after mango twig inoculation. (<b>C</b>) The images illustrate representative lesions elicited by the fungal strains 10 days after mango fruit inoculation. (<b>D</b>) Graphical representation of lesion areas on mango leaves measured as described in (<b>A</b>). (<b>E</b>) Graphic representation of lesion lengths on mango twigs measured as described in (<b>B</b>). (<b>F</b>) Graphic representation of lesion areas on mango fruits measured as described in (<b>C</b>). The data from 5 replicates are means ± SD with different letters that are significantly different (<span class="html-italic">p</span> < 0.05) among treatments using one-way ANOVA analysis. The control variable (CK) was inoculated with PDA (agar plugs) only.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Fungal Isolates, Growth Conditions, and Taxonomic Analysis
2.2. Extraction of dsRNA from Fungal Mycelium
2.3. Full-Length Analysis by Next-Generation Sequencing (NGS) Approach and RACE
2.4. Bioinformatic and Phylogenetic Analysis
2.5. Elimination of C. gloeosporioides Strain Ssa-44.1 dsRNA
2.6. Virus Transmission
Horizontal Transmission
2.7. Effect of Mycovirus on Fungal Colony Morphology, Growth, and Pathogenicity
2.8. Statistical Analyses
3. Results
3.1. Identification and Biological Characteristic of Virus-Infected Isolate Ssa-44.1
3.2. Genome Organization and Phylogenetic Analysis of Mycovirus Ssa-44.1
3.3. Biological Properties of CgRV1-Ssa-44.1 on C. gloeosporioides
3.4. Transmission of CgRV1-Ssa-44.1 in C. gloeosporioides
3.5. Effect of CgRV1-Ssa-44.1 on Virulence of C. gloeosporioides
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
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Strains | Spore Counting (Spores/mL) | |||
---|---|---|---|---|
1 | 2 | 3 | Mean ± SD * | |
Ssa-44.1 | 2.58 × 107 | 1.85 × 107 | 2.38 × 107 | 2.27 ± 0.38 × 107 a |
Ssa-44.1#18 | 4.25 × 108 | 5.39 × 108 | 4.95 × 108 | 4.86 ± 0.57 × 108 b |
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Suharto, A.R.; Jirakkakul, J.; Eusebio-Cope, A.; Salaipeth, L. Hypovirulence of Colletotrichum gloesporioides Associated with dsRNA Mycovirus Isolated from a Mango Orchard in Thailand. Viruses 2022, 14, 1921. https://doi.org/10.3390/v14091921
Suharto AR, Jirakkakul J, Eusebio-Cope A, Salaipeth L. Hypovirulence of Colletotrichum gloesporioides Associated with dsRNA Mycovirus Isolated from a Mango Orchard in Thailand. Viruses. 2022; 14(9):1921. https://doi.org/10.3390/v14091921
Chicago/Turabian StyleSuharto, Aditya R., Jiraporn Jirakkakul, Ana Eusebio-Cope, and Lakha Salaipeth. 2022. "Hypovirulence of Colletotrichum gloesporioides Associated with dsRNA Mycovirus Isolated from a Mango Orchard in Thailand" Viruses 14, no. 9: 1921. https://doi.org/10.3390/v14091921