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. 2013 Jan 22;110(4):1452-7.
doi: 10.1073/pnas.1213755110. Epub 2013 Jan 7.

Extracellular transmission of a DNA mycovirus and its use as a natural fungicide

Xiao Yu  1 , Bo Li, Yanping Fu, Jiatao Xie, Jiasen Cheng, Said A Ghabrial, Guoqing Li, Xianhong Yi, Daohong Jiang
Affiliations

Extracellular transmission of a DNA mycovirus and its use as a natural fungicide

Xiao Yu et al. Proc Natl Acad Sci U S A. .

Abstract

Mycoviruses are thought not to be infectious as free particles and to lack an extracellular phase in their life cycles, limiting the broad use of hypovirulence-associated mycoviruses in controlling fungal disease. Here, we demonstrate that purified particles of a DNA mycovirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), are infectious when applied extracellularly to its host Sclerotinia sclerotiorum. Virus particles isolated from an infected host can infect the hyphae of virus-free S. sclerotiorum directly when applied to hyphae grown on potato dextrose agar or sprayed on leaves of Arabidopsis thaliana and Brassica napus, regardless of vegetative compatibility affiliation. When applied to leaves, the virus can suppress the development of lesions. SsHADV-1 can also reduce disease severity and enhance rapeseed yield significantly under field conditions. SsHADV-1 has a narrow host range; it can infect Sclerotinia minor and Sclerotinia nivalis, sister species of S. sclerotiorum, and cause debilitation of these two fungi, but cannot infect or transfect other tested fungi, such as Botrytis cinerea, which shares the same family with S. sclerotiorum. Virus particles are likely to be very stable on the leaves of A. thaliana plants because viral DNA could be detected at 15 d postinoculation on unwounded leaves and at 10 d postinoculation on wounded leaves, respectively; however, this virus could not infect and move in plant cells. Our findings may prompt a reconsideration of the generalization that mycoviruses lack an extracellular phase in their life cycles and stimulate the search for other DNA mycoviruses with potential use as natural fungicides.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Infection of S. sclerotiorum with purified particles of SsHADV-1 following extracellular application to intact hyphae on PDA plates. (A) Sectoring in a colony of strain Ep-1PNA367 could be observed at 2 dpi; sectors are indicated by arrowheads. Strain Ep-1PNA367 was grown on a PDA plate for 1 d, and then virus particles were added to the PDA medium around the margin of the young colony, but not to contact it. PBS buffer was used as a control. The photographs were taken at 4 dpi. (B) Colony morphology of newly infected strain Ep-1PNA367; colony morphology of the virus-free stain Ep-1PNA367 and the virus-infected strain DT-8 are also shown. All cultures were incubated on PDA medium for 7 d at 20 °C. (C) Electrophoretic profiles of genomic DNA extracted from the cultures shown in B are shown. DNA samples were fractionated on 1.0% agarose gel; the positions of host genomic DNA and viral DNA are indicated to the right. Lane M: λ-Hind III-digested DNA marker. Lanes 1–3, DNA samples of isolates Ep-1PNA367, virus infected Ep-PNA367, and strain DT-8, respectively.
Fig. 2.
Fig. 2.
Protection of plants against S. sclerotiorum using SsHADV-1 particles. (A) Virus particles prevented S. sclerotiorum from infecting and killing plants of A. thaliana. Viral particles at different concentrations were spread on leaves (only one leaf was sprayed for each plant), and mycelial agar plugs were placed on the treated leaves. The fungal pathogen-inoculated plants were kept in an incubator with high humidity (100%) at 20 °C, and photographs were taken at 6 dpi. (B) Therapeutic activity of SsHADV-1 against the attack of S. sclerotiorum. Crude preparation of virus particles inhibited the expansion of S. sclerotiorum on rapeseed plants and cured the lesions. S. sclerotiorum mycelial agar plugs were used to inoculate plant leaves, leaves were incubated for 2 d for lesion development, and then viral particles preparation, Carbendazim (100 ppm) or PBS buffer, was sprayed on plants at 2 dpi and 3 dpi, respectively. Three leaves were inoculated for each plant. Plants were maintained in an incubator with high humidity (100%) at 20 °C, and photographs were taken at 8 and 12 dpi.
Fig. 3.
Fig. 3.
Infection of protoplasts from two Sclerotinia species (S. minor and S. nivalis) closely related to S. sclerotiorum with purified SsHADV1 particles. (A) Colony morphology of virus-infected and wild-type strains of S. minor and S. nivalis. Strains were incubated at 20 °C for 5 d. (B) Viral genomic DNA was extracted from newly infected strain 3-4-1 of S. minor and strain Let-19 of S. nivalis. Viral genomic DNA is indicated. Lane M: DL2000 DNA marker.
Fig. 4.
Fig. 4.
Detection of SsHADV-1 on plant leaves and in plant cells. (A) Detection of SsHADV-1 on leaves of A. thaliana using specific primers designed based on viral sequence. Viral particles were spread on leaves with a brush or with a brush and quartz sand. After 0, 3, 5, 7, 10, 15, and 30 d, inoculated leaves, noninoculated leaves, and newly emerged leaves on inoculated plants were collected and subjected to total DNA extraction for PCR amplification of viral genomic DNA. (B) FISH analysis of SsHADV-1 replication in cells of inoculated leaves of A. thaliana. Fluorescence photomicrographs of plant cells and virus-infected fungal compartments of strain Ep-1PNA367 after FISH with biotinylated CP probes. Hybridization signals of the CP probe were detected using Cy3-labeled streptavidin. DAPI staining was used as a control. The fluorescence in plant cells or fungal compartments was examined with a fluorescence microscope (Nikon 80i); the blue signals were examined at 360 nm and the red signals excitation at 550 nm. (Scale bar: 20 μm.)
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