Wilmer J. Cuellar

UNRAVELING VIRUS COMPLEXES IN PLANTS

2015Palmira, 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

AFRI

CAAM

ERIC

A

Mixed infected Single infected

In the search of ‘Frogskin’ aethiologyIt was first reported in 1971 from southern Colombia (Pineda, et al. 1983)

Cauca1971

• 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

AsiaCsGMV 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 19FSD23

Mcol2215 1

6FSD5

SM909-25 Mcol2737-7s

546010-113FSD86

FSD80 OUT

87

6960

88

70

62

7360

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

% o

f inf

ectio

n

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 UsetINTA, 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

61100

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 p7Rest 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.