Pl Path 502-Viroids - CSK Himachal Pradesh Agricultural University

Viroids
Pl Path 502
Dr. PN SHARMA
Department of Plant Pathology
CSK HP Agricultural University
Palampur-176 062 (HP State) INDIA

Developments in molecular biology of the 20 th 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

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
Yellow green rods denote the first
viroid as seen in electron micrograph
Therefore often denoted as subviral
particles or agents
THEODOR O. DIENER
Discoverer of the viroid 1971

• 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
DISEASE
FIRST
REPORT
COUNTRY
LOSS
VIROID
ETIOLOGY
VIROID
NAME
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

• RNA, Low molecular weight 0.8-1.3x10 5 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
Disease First report Etiology
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

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
Eradication of Sources of inoculum
Cultural Practices
Quarantine Regulations
Biotechnological Approach
Biotechnological Approach