Dr. PN SHARMA

Department of Plant Pathology

CSK HP Agricultural University

Palampur-176 062 (HP State) INDIA

Pl Path 502

Viroids

Developments in molecular biology of the 20th century

Discovery of double helical DNA

Cracking of genetic code

Development of recombinant DNA and PCR

techniques

Elucidation of 3D protein structure

Viroids and Prions – molecules at the threshold of

origin of life

THEODOR O. DIENER

Discoverer of the viroid 1971

Yellow green rods denote the first viroid as seen in electron micrograph

Therefore often denoted as subviral particles or agents

Viroids (T.O. Diener, 1971): are small, low mol

wt. RNA units (250-370 bp.), lack protein coat, replicate themselves and cause disease Example: Potato spindle tuber viroid, coconut codang-cadang.

Autonomously replicating Pathogens, unencapsidated Single

Self replicating circular, low molecular weight RNA without protein coat

Infect only plant cells

Produce variable symptoms on different hosts like stunting, bark scaling, proliferation, veinal necrosis and also symptom less carrier (No symptoms)

Vegetatively propagated, highly seed and pollen transmitted

Losses caused byviroid diseases

Potato Spindle 1917 USA 26 - 90% 1971 PSTVd

Tuber USSR 54%

China 60%

Canada 64%

Cadang Cadang 1927 Philippines 20 million 1975 CCCVd

of coconut nuts

Hop Stunt 1952 Japan 17 - 60% 1977 HSVd

DISEASE LOSS VIROID

NAME COUNTRY

FIRST

REPORT

VIROID

ETIOLOGY

RNA, Low molecular weight 0.8-1.3x105 D

Single stranded - 246-375 nucleotides

Circular forms with secondary structure - Highly base paired

Rich in G+C Content

To date sequences of 25 viroids and 160 viroid variants are available in gene databases

Model of viroid domain T1 and T2; Terminal Domains, P; Pathogenicity Domain, V; Variable Domain and

C; Central Conserved Domain

Self Replicating -

Auto cleaving -Due to Presence of Ribozymes

By rolling circle mechanism

No translation

Ribozymes are catalytic RNAs with intrinsic ability to break and form covalent bonds. They cleave RNA in 2 fragments with 5’ hydroxyl and 2’ – 3’ cyclic phosphate in a non hydrolytic reaction. The process is often referred to as catalytic cleavage

ROLLING CIRCLE MECHANISM

Asymmetric model Symmetric model

Common in plasmid or bacteriophage DNA and the circular RNA genome of e.g.

Viroids, and DNA viruses e.g. geminiviruses

Rolling circle DNA replication is initiated by an initiator protein encoded by the plasmid or

bacteriophage DNA, which nicks one strand of the double-stranded, circular DNA

molecule at a site called the double-strand origin, or DSO.

The initiator protein remains bound to the 5' phosphate end of the nicked strand, and the free 3' hydroxyl end

is released to serve as a primer for DNA synthesis by DNA polymerase II.

Using the unnicked strand as a template, replication proceeds around the circular DNA molecule, displacing

the nicked strand as single-stranded DNA. Displacement of the nicked strand is carried out by a host-encoded

helicase called PcrA (plasmid copy reduced) in the presence of the plasmid replication initiation protein.

Continued DNA synthesis can produce multiple single-stranded linear copies of the original

DNA in a continuous head-to-tail series called a concatamer.

These linear copies can be converted to double-stranded circular molecules through the

following process:

First, the initiator protein makes another nick to terminate synthesis of the first (leading) strand. RNA

polymerase and DNA polymerase III then replicate the single-stranded origin (SSO) DNA to make another

double-stranded circle. DNA polymerase I removes the primer, replacing it with DNA, and DNA ligase joins

the ends to make another molecule of double-stranded circular DNA.

Plant Appeareance

Stunting/ dwarfing; Proliferation leading to bunching

Symptomless / latent

Leaf

Epinasty, venial necrosis, yellow/corky vein, puckering

Stem

Bark scaling/ splitting particularly at bud union region.

Stem discolouration

Flower

No symptoms, no sterility

Fruit/ Seed

Rough skin, scar skin

Sap Tomato bioassay

Graft Citron bioassay, Cucumber bioassay

Vegetative Pruning / Cutting Knives

Seed Very high rate

Pollen High rate

Insect Not yet confirmed universally

Symptoms on Inoculated Tomato Spindle Shaped Tubers

Artificially inoculated seedling (left), 6

years after inoculation, showing stunting,

sterility and disordered pinnae, compared

with a healthy seedling.

Severe infection leading to tree decline

Stunting

CEVd CEVd-t

Intensity of disease known only

after deformation of fruit is

observed

Symptoms on leaves appear as mild

chlorosis

Viroid Diseases in India

Citrus exocortis 1968 1992

Tomato Bunchy top 1982 1989,1992

Potato spindle tuber 1989 1991

Tobacco Proliferation 1991 1991 Coleus Symptomless 1991 1992

Citrus latent 1991 1992

Apple Scar Skin (Dapple) 1995 1995

Citrus yellow corky vein 1974 1996

Disease First report Etiology

VIROID INFECTIONS IN DIFFERENT

PLANT FAMILIES

ASTERACEAE : CCMVd, CSVd (2)

CARYOPHYLLACEAE: CSVd (1)

CUCURBITACEAE: CPFVd (1)

GESNERIACEAE: CLVd (1)

LABIATAE: CYVd, CbVd (2)

LAURACEAE: ASBVd (1)

PALMAE: CCCVd, CTVd, OPFYVd (3)

ROSACEAE: ASSVd, PLMVd, PDVd, PBCVd (4)

RUTACEAE: CEVd, HSVd (citron), CiVVd, CIT.

CACHEXIA (4)

VITACEAE : HSVd (gv), HSVd (ggv), AGVd,

GYSVd, G 1bVd , HSVd (hop), HLVd,

CEVd (gv) (8)

SOLANACEAE: PSTVd, ITBTVd, TASVd,

TAPMVd, NgPVd (5)

THEACEAE: TDDVd (1)

primary sources of inoculum: Seed and pollen

Vegetative propagation

Large scale monoculture

Escape from natural host to commercial crop and

vice-versa

Evolution of natural recombinants

Lack of adequate quarantine check

Detection of Viroids

Detection of Citrus Viroids by PCR

L-R: Marker (100bp), CEVd (2,3), CVd II(5),

CVd gr III (6,7), CYCVVd (9,10)

Management of viroid diseases

Biotechnological Approach

Eradication of Sources of inoculum

Cultural Practices

Quarantine Regulations

Biotechnological Approach