Development of Mycovirus Causing

Hypovirulence to Phytopathogen
as Biocontroller

Hiromitsu Moriyama

Laboratory of Molecular Cell Biology, Department of

Applied Biological Sciences, Tokyo University of
Agriculture and Technology
 Fig.1 Mycovirus of fungi

“Fungi having genome of double-stranded RNAs are collectively called Mycovirus.”

Latency

Giving Latent Mycovirus

attenuation Mycovirus of Growth restricting
（Attenuation） factor

・Most of mycovirus do not influence host-fungi clearly.

・Some mycoviruses play a role in restricting growth of host-
fungi and attenuating pathogens.
 Mycoviruses associated with host virulence modulation

Spices Family Nucleic Acid Host (disease)

Cryphonectria parasitica Hypoviridae dsRNA C. parasitica
hypovirus (9-13kbp) (Chestnut blight）
Helminthosporium victoriae dsRNA H. victoriae
Totiviridae
virus 190s (5kbp) (Victoria blight of oats)
Helminthosporium victoriae Chrysoviridae Four dsRNA H. victoriae
virus 145s (2.8-3.6kbp) (Victoria blight of oats)

Unclassified virus dsRNA Rhizoctonia solani

(3.6kbp) (Rhizoctonia disease of potato)

Ophiostoma novo-ulmi ssRNA O. novo-ulmi

Narnaviridae
mitovirus (about 2.5kbp) (Dutch elm disease)

Mycoviruses related to host fungal virulence modulation were screened

in several strains of Alternaria alternata or Magnaporthe oryzae.
 Target in middle term：Research and nature analysis of
mycoviruses to restrict Magnaporthe oryzae and A.Alternate.

Diseased tissues on an
oryza sativa leaf Magnaporthe oryzae A.Alternate Soil or
fruits, vegetables
Separation Seperation

Poor growth of Hyphae Poor growth of Hyphae

Isolation from stock Isolation from stock
cultures of fungi cultures of fungi
Mycovirus
（Double-stranded RNA genome）
Mycovirus
（Double-stranded RNA genome）

Target in result 1. Identification of mycovirus to attenuate host pathogen

2. Identification of genetic structure and pysical
chemical properties of the virus
Mycovirus containing four double-stranded RNAs
affects host fungal growth in Alternaria alternata.
 Detection of dsRNAs in Alternaria alternata
A Total nucleic acids treated with B CF-11 column purified
DNaseⅠ＆S1Nuclease dsRNA
M 1 2 3 4 5 M 5

19.3
19.3 Undigested
DNA 6.2
7.7 4.3
6.2 dsRNA 1
4.3 3.5 dsRNA 2
3.5 2.7 dsRNA 3
2.7
1.9
1.9 1.5 dsRNA 4
1.5
(kbp)
(kbp)

M. DNA marker 1. A. alternata (12L-5)

2. A. alternata (22L-5) 3. A. alternata (15F-3)
4. A. alternata (O-159) 5. A. alternata (EGS 35-193)

Various sized (from 1.5 to 6.2kbp) dsRNAs were detected in A. alternata.

 Influence of dsRNAs on phenotype of A. alternata
① EGS 35-193; Total RNA
original strain （dry weight 5mg）
① ②
• late growth
• reduced number of aerial mycelia
19.3 Undigested
• irregular pigmentation
• detection of four dsRNAs 7.7 DNA
6.2
Expose to 1μg/ml Cycrohexamaide 4.3 dsRNA 1
Cure-
Hyphal tip cultivation (Repeated) 3.5 dsRNA 2
treatment dsRNA 3
2.7

② E118; 1.9 dsRNA 4

dsRNA cured strain 1.5
• normal growth (kbp)
• large number of aerial mycelia
• normal pigmentation ① EGS 35-193
• lower concentration of dsRNAs ② E118

High copy number of viral dsRNAs caused phenotypic changes

on their host, A. alternata.
 Virus-like particles of A. alternata mycovirus
Silver stain of SDS-PAGE
Electron micrograph of virus-like particles of virus preparation
LMW HMW 1 2
Bar = 100 nm

212
170 dsRNA

116 RdRp
94 CP

76
67
Bar = 100 nm

Purified virus preparation was stained (kDa)

with 2 % uranyl acetate
1. Virus preparation
2. Purified virus
Isometric virus particles with a diameter of about 33 nm were observed.
 Genome organization of A. alternata mycovirus
Single open reading frame (1149 a.a. )
dsRNA 1
RdRP_4 conserved domain A (40-50 nt)
(3,567 bp) 48 69

Single open reading frame (844 a.a. )

dsRNA 2
A (40-50 nt)
(2,744 bp) 53 157

Single open reading frame (759 a.a. )

dsRNA 3
A (30-40 nt)
(2,540 bp) 52 209

Single open reading frame ( 393 a.a. )

dsRNA 4 A(20-30nt)
(1,385 bp) 53 150

RdRP_4 ：Viral RNA-directed RNA-polymerase

This family includes RNA-dependent RNA polymerase proteins (RdRPs) from
Luteovirus, Totivirus and Rotavirus.

dsRNA 1 encodes a single open reading frame that contains

a Totivirus-like RdRP (RdRP_4) conserved domain.
 Phylogenetic tree of the RdRp regions of
A. alternata mycovirus and selected mycoviruses
98 GLV-L1

97 SsRV-2
Hv190SV
94
49 SsRV-1
Totiviridae
32
LRV-2-1
ScV-L-A
68
98 ScV-L-BC
69 Hv145SV
100 PcV
Chrysoviridae
67
Cy-aV

A. alternata
AaV mycovirus
AhV
97 FsV-1
Partitiviridae
HuRV-C Reoviridae
0.1

The A.alternaria mycovirus is related to Crysoviridae, Partitiviridae,

Totiviridae.
 Comparison between AaV and
i mycovirus in three families

Virus family dsRNA genome

Virus particle 3’ poly(A)
(virus name) Segment Size(kbp)

AaV 4 1.5-3.6 Isometric, 33nm 20-50 nt

Totiviridae 1 4.6-6.7 Isometric, 33-40nm Not found

Partitiviridae 20-30 nt
2 1.4-2.2 Isometric, 30-38nm
(Interrupted)

Crysoviridae 4 2.4-3.6 Isometric, 35-40nm Not found

Conclusion
AaV seems to be related to the typical mycovirus families, totiviridae,
partitiviridae and crysoviridae, but not classified into these families.
Mycoviruses Associated with Impaired
Growth of the Rice Blast Fungus,
Magnaporthe oryzae
 Comparison of phenotypes between mycovirus
-Free and -Infected strains, Magnaporthe oryzae
Free Infected Infected Infected
MOT 40 3.6 MOS
2121 3.6 MOS 22 3.6 MOT 25
kbp kbp
40 kbp

Free Infected Infected Infected

3.6
MOS 57 3.6 MOT 27 kbp
MOT 29 3.6 MOT 38
kbp kbp

Mycovirus-infected strains showed abnormal growth phenotypes.

 Isolation of viral dsRNAs from M. oryzae
Methods:
1. Extraction of whole nucleic acids from fungal mats,
then digestion with DNase I and S1 nuclease.
2. CF-11 column chromatography is sometimes performed.

Inoculate Harvest SDS-Phenol method

Enzyme digestion

PDA medium Agarose gel electrophoresis

at 24 ℃ for 2weeks YG liquid medium
at 24 ℃ for 2weeks “dsRNA carring or not”
 Comparison of growth rates between the
mycovirus-infected strains and the free strains
MOS 9 Mycovirus-free

Growth Rate
5
6 days 10 days (cm) 4.5
4

Elongation
of Mycelia
3.5
MOT 22 Mycovirus-infected 3
2.5
2
1.5
1
0.5
0
0 1 2 3 4 5 6 7 8 9 10
6 days 10 days
Cultured Days
MOT 27 Mycovirus-infected
Free MOS 9
Infected MOT 22
Infected MOT 27

6 days 10 days

Mycovirus-free strains showed fast growth rates than the infected strains.
 Phenotypes of mycovirus-Free and -Infected strains

Free strain

・ Fast growth
・ Regularly radial mycelia growth
・ Normal pigmentation

MOT 44（Surface） MOT 44（Reverse）

Infected strain
・Slow growth
・Irregular mycelia growth
・Irregular pigmentation
・Unusual aerial mycelium
・Autolysis
MOS 48（Surface） MOS 48（Reverse）
 Phylogenetic analysis of the RDRP conserved motifs
of MoCV1, 2 and selected totiviruses and partitiviruses

100 M. oryzae chrysovirus-1

48 M. oryzae chrysovirus-2
100
A. mycovirus 1816
46 A. bisporus virus 1 Chrysoviridae
A. cherry disease associated virus
92 H. victoriae 145S virus
73 P. chrysogenum virus
U. maydis virus H1
39
S. cerevisiae virus L-A
77
M. oryzae virus 2 Totiviridae
99 M. oryzae virus 1
56 H. victoriae virus 190S
H. annosum virus
100
A. hypoxylon virus
Partitiviridae
97 F. poae virus 1
99 R. solani virus

0.2
 Final Target：Establishment of mycovirus infection method
and preparation of stock cultures of fungi
“Giving
attenuation”

Giving Infection
Toxic Magnaporthe oryzae
attenuation
“Virus infection”

Attenuated Magnaporthe oryzae “Settlement”

Utilization as biocontrol agents having “vaccinal effect” to toxic stock cultures of fungi