Wilmer J. Cuellar
UNRAVELING VIRUS
COMPLEXES IN
PLANTS
2015
Palmira, Colombia
E-mail w.cuellar@cigar.org

Viruses
A virus is basically a protein shell with a few
genes inside.
They occur as part of fast evolving and complex
communities and they are ecologically
important components of the environment.
It is not longer practical for one laboratory to
focus on single strains grown in pure culture.
They are historically associated with disease.

Mosaics Yellowing
Ring SpotLeaf Curling
Most of plant
pathology in the
early 1900s
consisted of
symptom
description
In the beginning we had… ‘mosaics’,
‘yellowings’, ‘ringspots’, etc.

TBT Early plant virology days

‘This masking of symptoms or development of
immunity, or whatever it is, seems to hold under
greenhouse conditions for practically all the plants
tested.’
‘Attempts have been made to produce symptoms on
these leaves from other plants, but without success
(...). The virus is still present even though the disease
symptoms fail to appear.’
Wingard, 1928. J. Agric. Res. 37, 127-153.
But if you leave the plants to grow older…

a.k.a. ‘immunization’, ‘Protective inoculation’, ‘interference’, ‘reciprocal
protection’, ‘mutual antagonism’, ‘prophylactic inoculation’, etc. Worked
with similar (related) viruses and under laboratory conditions.
McKinney, 1929. Mosaic diseases in the Canary Islands, west Africa, and Gibraltar. J. Agric. Res. 39, 557-578.
Salaman, R.F. 1933. Protective inoculation against a plant virus. Nature. 131, 468-468.
You will get: Cross protection

Things are different in nature: latent
and mild viruses
In 1925 Johnson (US) analyzed healthy-looking potatoes and found a ‘mild mottle
virus’ in almost all US potato varieties (Potato virus X).
The X virus was also found in Europe and described as ‘simple mosaic’ in some
varieties or ‘latent virus’ in others.
In nature:
Johnson J. 1925. Wisc. Res. Bull. 63:12pp.
Smith K. 1931. Proc. Royal Soc London. 109, 251-267.
Salaman RF. 1937. Proc. Royal Soc London. 229, 137-217.
Dykstra T.P. 1938. Phytopathology. 629:40-67.
X + YYX

Mixed infections are common in
cassava worldwide
CBSD mosaic CMD mosaic
Caribbean mosaicCommon mosaic Frogskin mosaic
No mosaic
AFRICAAMERICA
Mixed infected Single infected

In the search of ‘Frogskin’ aethiology
It was first reported in 1971 from southern Colombia (Pineda, et al. 1983)
Cauca
1971
• In 1980, flexous viral particles were observed in cassava plants displaying FSD
(Pineda B., et al., 1980)
• In parallel, mycoplasma related structures were found. Complex infection
virus-mycoplama (Pineda B. & Lozano J.C., 1981)
• The landrace Secundina (MCol 2063) expressed mosaic symptoms where FSD
was endemic (CIAT Cassava Program Annual Reports, 1984,1985)
• The presence of a potexvirus (CsVX?) in endemic regions of FSD was reported
(Harrison B. D. & Lennon, A. M., 1989)
• Multiple dsRNA (viral?) were associated to FSD (Cuervo M., 1989)
• In 2007, it is reported the a phytoplasma in FSD-affected plants (Alvarez E., et. al.
2009).
• In 2008, a REOvirus is found with cassava plants affected by FSD (Calvert L., et. al.
2008).
• FSD plants giving negative results to REOvirus and to Phytoplasma
• In 2014 we showed that all affected plants contain a complex infection and
there was not enough evidence to associate previous pathogens separately
(Carvajal-Yepes et al., 2014)

Sites of study: North Coast, Valle del Cauca, Eastern Plains
Improving diagnostics to reduce risk
-e.g.Cassava

H RS H RS H RS
Symptoms vary depending on the
root variety
Venezolana
(MCOL-2215)
Mtai8 MCOL-2737

Reproducing motling symptoms
infectedControl

Reproducing leaf deformation
symptoms
infectedControl

Updated list of virus species infecting
cassava
NAME TAXONOMY DIAGNOSTIC
America
CsCMV Potexvirus ELISA/RT-PCR
CsVX Potexvirus ELISA
CsNAV Potexvirus RT-PCR
CsVMV Cavemovirus PCR
CsFSaV Reoviridae RT-PCR
CsPLV Polerovirus RT-PCR
CsTLV Torradovirus RT-PCR
Asia
CsGMV Nepovirus RT-PCR
ICMV Geminvirius PCR
SLCMV Geminivirus PCR
Africa
ACMV Geminivirus PCR
EACMV Geminivirus PCR
SACMV Geminivirus PCR
ICVM Geminivirus PCR
CBSV Ipomovirus RT-PCR
UCBSV Ipomovirus RT-PCR
Let’s take a look
Cassava Virologists

Phylogenetic
tree (nt)
CM4919-1
Sec94-2
Sec92-1
CM523-7
CM6740-7_1
CM4574-1
Sec92-2
CM6740-7
CM6740-7_1
CM6740-7_2
CM6740-7
CM4919-1
CM4574-7
Sec13
Nataima
Lyophilized 19
FSD23
Mcol2215
1
6
FSD5
SM909-25
Mcol2737-7s
546010-113
FSD86
FSD80
OUT
87
69
60
88
70
62
73
60
0.01
in-vitro
Field collected
Improving diagnostics tools by
surveying virus diversity
AAAAAA
AAAAAARNA1
RNA2
Full genome characterization CsTLV

Tools for evaluating virus cleaning protocols
Positive Negative
%ofinfection
N: 246
CsFSaV
CsFSaV: 42%
CsPLV: 4.7%
CsTLV: 4.7%
5.1 5.2 5.3 5.4

To detect early build up of viruses in
the field
Virus free plants became infected
in the first cycle45%
60%

Tools to evaluate viruses in roots
Transgenic Line L17Transgenic Line L10
H
VIRUS 2VIRUS 1
NT H
VIRUS 2VIRUS 1
NT
H
LEGEND
VIRUS 1 and VIRUS 2: Different virus ifnections.
H: Transgenic line grafted with not-infected ‘Secundina’.
NT: Non-transgenic line.

Tools to identify their pathogenicity proteins
important in disease
AAAAAA
Normal light Ultraviolet light
1 2 3
C 1
2 3

Three examples of reinforcing
diagnostics

Cassava common mosaic disease in the
South: Re-emerging mixed infections
Photos: Ovidio Antonio Uset
INTA, Misiones, Argentina
CsCMD-2014 Reports:
Misiones (Arg)
Corrientes (Arg)
Itapúa (Par)

CsCMV: Early identification, early
response
Cordoba-COL
PRO29-COL
GU09-COL
Arg24-4
Corrientes-ARG
Mcol1505-1
U23414-Brazil
Mcol22-1
Arg126-6
Arg120-3
Arg128-1
Arg104-3
Arg22-1
Bra456-4
Par92-1
Arg25-1
Arg28-4
Arg97-4
JF913280-Parana
Arg127-1
Arg113-6
Arg121
Arg34-4
PAr94-2
Outgroup
99
94
95
100
82
61
100
71
77
70
98
100
91
0.05
2013-2014

p22 can suppress
the defense of
the plant
p22 enhances the
accumulation of the
virus
p22 by itself
Induce severe
symptoms in
other plants
(-) (+) (-) (-)
P-Pro MTR HEL RdRp p26 p7 p22
Uganda
Sweetpotato: Sometimes viruses
change strategies
P-Pro MTR HEL RdRp p26 p7
Rest of
the
world
East Africa strain
West Africa strain

Citrus: Re-evaluation of cleaning
systems
Main citrus regions in Colombia (2014)
17 ICA-registered nurseries
5 comercial greenhouses
80 samples
99% infected by known pathogens (Mixed infections)
CEVd 46%
HSVd 81%
CTV 60%

Main drivers of disease
Anderson PK., et al. 2004. Emerging infectious diseases of plants (...) TRENDS in Ecol and Evol. 19:535-544.
Early identification should include the survey for potential pathogens before
they jump into crops.

Take home messages and Future
directions
A high diversity of hosts implies a even wider biodiversity of potential
pathogens. We need to understand the biological significance of that
diversity and their interactions.
It is not longer practical to focus on diagnosis of single isolated pathogens
grown in pure culture, especially with diseases affecting RTB crops.
An “early identification, early response” strategy. Emphasis basic field
research and biological tests. Sequences are needed but not enough.
Including the diversity of potential pathogens in wild plants.
We suggest the establishment of a Surveillance Network which should
incorporate information on disease-conductive environments and
considering historical information on previous epidemics.

Carvajal-Yepes M, Olaya C, Lozano I, Cuervo M, Castaño M, Cuellar WJ.
(2014) Unravelling complex virus infections in cassava. Virus Research 186,
76-86.
Legg J, Kumar L, Makeshkumar T, Ferguson M, Kanju E, Ntawuruhunga P,
Cuellar WJ. (2015) Cassava virus diseases: Biology, Epidemiology and
Management. Advances in Virus Research. 91, 85-142.
DiFeo L, Zanini A, Rodriguez P, Cuervo M, Carvajal-Yepes M, Cuellar WJ.
(2015) First report of Cassava common mosaic virus and Cassava frogskin
associated virus infecting cassava in Argentina. Plant Disease. 9, 733.
Van der Vlugt R, Verbeek M, Dullemans AM, Wintermantel WM, Cuellar WJ,
Fox A, Thompson JR. (2015) Torradoviruses. Annual Review of
Phytopathology. 53, 23.1-23.28.
Cassava Virology - Publications

Cassava virology – CIAT
Ivan Lozano Monica Carvajal Jenyfer Jimenez
MSc Thesis completed:
Cristian Olaya, 2014. MSc-Thesis. UNAL-Palmira, Colombia.
Ana M. Leiva, 2015. MSc-Thesis. UNAL-Palmira, Colombia.
THANK YOU FOR YOUR ATTENTION
Alejandro Quintero Bertha Garcia
We inherited a strong Research Laboratory, founded by Dr. Francisco Morales.