MSc

Papaya is an important fruit crop which is cultivated extensively both in tropics and
subtropics. During the last decade, the area under papaya cultivation has dramatically increased
due to the introduction of superior varieties. A major setback subsequent to the introduction of
such new varieties has been the incidence of papaya ringspot disease caused by Papaya ringspot
virus (PRSV) which leads to almost 100 per cent yield loss. Considering the importance of the
disease, the project was undertaken with the objectives of characterization of the virus, molecular
and immunodiagnosis along with disease management.
The research project was initiated with purposive sampling surveys conducted in different
papaya orchards of Thrissur district in order to document the symptoms under natural conditions,
to assess the incidence and severity of the disease and to collect infected samples for further
studies. The maximum disease incidence and disease severity recorded were 99.6 and 96.67 per
cent on papaya variety Red Lady from Vellikulangara and Puthur respectively. The development
of symptoms was studied under artificial conditions also through mechanical inoculation of
healthy papaya seedlings with virus inoculum maintained in insect proof net house conditions. The
salient diagnostic symptoms of the disease observed on leaves were chlorotic spots, mottling, vein
thickening, puckering, leaf distortion, shoestring symptom along with presence of oily streaks on
the petiole. The fruits of infected plants presented typical oily concentric or broken ringspots on
fruit surface along with malformation. Histopathological studies of infected leaves revealed
disruption of the epidermis, disorganization of parenchyma, disintegration of chloroplasts and
deposition of crystalline bodies.
The studies on virus transmission confirmed that it is transmitted through plant sap from
infected to healthy papaya plants. Seed transmission studies revealed that PRSV is not seed -borne.
Twenty one plant species including weeds seen in and around papaya orchards were tested for
studying the host range of the virus and only five plant species viz., Cucumis sativus, Cucurbita
moschata, Trichosanthes cucumerina, Momordica charantia and Chenopodium amaranticolor
developed symptoms after artificial inoculation of PRSV and thus proved to be the hosts of the
virus. Morphological characterization done using electron microscopy showed the presence of
typical flexuous rod particles of size 807.74 nm x 12 nm which indicated that the virus belongs to
genus Potyvirus and the etiology of the disease was confirmed as Papaya ringspot virus.
Immunodiagnostic technique was validated using Direct Antigen Coating - Enzyme Linked
Immuno Sorbent Assay (DAC-ELISA) and infected samples showed positive reaction to PRSV
antiserum and could be detected at 1:200 dilution of primary antibody and 1:10,000 dilution of
secondary antibody. Molecular characterization of PRSV was also carried out through Reverse
Transcription Polymerase Chain Reaction (RT-PCR). The nuclear inclusion b (NIb) gene and the
coat protein (CP) gene were amplified using reported primer pairs which yielded amplicons of
approximate size of 1700 bp. The PCR products were outsourced for sequencing and in silico
analysis of the sequences obtained revealed that the isolates of present study are more similar to
Calicut isolate of PRSV, PRSV- Ca (DQ666640.1)
A pot culture experiment under insect proof conditions was also conducted to evaluate the
effects of selected botanicals, chemicals and biocontrol agents for disease management. Among
the fourteen treatments, Bougainvillea leaf extract, 10 per cent (T7) was the most effective with
lowest disease severity (6.67%) followed by foliar spray and soil drenching with Pseudomonas
fluorescens 2 per cent (T3) with a disease severity of 11.11 per cent as against 97.77 per cent
recorded in untreated control plants. Plant height recorded after each treatment application
revealed that both 10 per cent Bougainvillea leaf extract (T7) and 2 per cent P. fluorescens are
equally superior to all other treatments. However, with respect to mean girth of stem, maximum
value (2.91 cm) was recorded in P. fluorescens (T3) The data on virus titre of all treated plants
assessed through DAC-ELISA revealed that the concentration of virus particles was minimum in
plants treated with T7 and T3.
The outcome of this study would facilitate early detection and elimination of source of
virus infection and thereby prevent the spread of disease in the field. The information generated
on molecular characterization of PRSV isolates under could be applied in genetic engineering. The
project also revealed the potential of botanical, Bougainvillea spectabilis leaf extract and
biocontrol agent, Pseudomonas fluorescens for the ecofriendly management of papaya ringspot
disease.