Ph.D

The research work entitled “Molecular diagnosis and management of Papaya
ringspot virus causing papaya ringspot disease” was undertaken in the Department of
Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram, during
2019-24, with the objectives; molecular diagnosis and recombinant coat protein
production of Papaya ringspot virus (PRSV), and evaluation of the efficacy of
beneficial microorganisms and botanical inthe management of papaya ringspot
disease (PRSD).
Roving survey was carried out across five Agro-ecological units (AEUs) of
Kerala. The disease incidence (DI) ranged from 50.25 per cent (Kayyur-Cheemeni) to
100 per cent (Kalliyoor, Venganoor, Balaramapuram, Pallichal, Kayamkulam,
Mavelikkara, Velukkara, Irinjalakuda and Shoranur). Vulnerability index (VI) of the
plants to PRSV in the surveyed locations ranged from 33.54 (Badiyadkka) to 98.22
(Kalliyoor). Serological and molecular detection confirmed the presence of PRSV in
all the 20 symptomatic samples collected during survey. Phylogenetic tree constructed
with the deduced amino acid sequences of CP gene of 11 Kerala PRSV isolates,
revealed that Thiruvananthapuram isolates clustered together, indicating their
relatedness during evolution.
Mechanical inoculation on two months old Red Lady papaya seedlings under
insect proof conditions was carried out to identify the most virulent PRSV isolate
collected from different AEUs. At three months after inoculation (MAI), Kalliyoor
isolate exhibited highest VI (96.63) followed by Alur (95.48) and Venganoor (95.22)
isolates. The lowest VI was observed in Kayyur-Cheemeni isolate with 57.95 VI,
followed by Cherpulassery (60.58).
The recombinant coat protein of PRSV was induced in pLATE 31 expression
vector with C terminal histidine tag, within BL21(DE3)pLysS expression host. PRSV
recombinant coat protein was purified using Ni-NTA column chromatography. A
single band was observed at 35 kDa in SDS-PAGE analysis and western blotting with
PRSV antiserum, confirmed the presence of purified recombinant coat protein in the
soluble fraction.
Pot culture experiment was conducted to evaluate the management of PRSD
using Piriformospora indica. The initial colonization of P. indica inside the papaya
roots was observed five days after germination of the seeds grown in P. indica massmultiplied
medium. Prophylactic colonization of P. indica exhibited lowest VI
(23.10) and 68.27 per cent reduction in VI over control diseased plants at five months
after PRSV inoculation (MAI). Double antibody sandwich - Enzyme linked
immunosorbent assay (DAS-ELISA) at 5 MAI revealed lowest absorbance (0.23)
indicating lowest virus titre in P. indica pre-colonized plants upon PRSV inoculation,
compared to control diseased plants (1.23).
The accumulation of reactive oxygen species (ROS) i.e., H2O2 and superoxides
in the leaves were analyzed using DAB (diaminobenzidine) and NBT (nitro blue
tetrazolium chloride) staining respectively. P. indica-colonized plants upon PRSV
inoculation indicated a higher initial accumulation of ROS at three weeks after
inoculation (3 WAI) and further reduction at 6, 9 and 12 WAI of PRSV, compared to
control diseased plants. P. indica-colonized plants also exhibited enhanced
antioxidant defence enzyme activity viz., catalase, peroxidase, polyphenol oxidase,
phenylalanine ammonia lyase, glutamate synthase and superoxide dismutase
compared to control diseased plants. Amongst PRSV inoculated treatments, P. indica
pre-colonized plants exhibited highest number of leaves (20.86), leaf area (365.14
cm2), plant height (108.29 cm), stem girth (7.26 cm), shoot biomass (423.43 g), root
biomass (133.96 g) and chlorophyll content (2.84 mg g-1 of fw) at five months after
transplanting (5 MAT).
Effect of P. indica, Bougainvillea spectabilis leaf extract (10 %) and
Pseudomonas fluorescens (2 %) were evaluated against natural incidence of PRSD
under field conditions at Instructional farm, College of Agriculture, Vellayani and
Coconut Research Station, Balaramapuram, Thiruvananthapuram. P. indica-colonized
plants exhibited lowest VI (37.63), with highest reduction in VI over control (53.85
%) followed by B. spectabilis treated plants (37.49 %) and P. fluorescens treated
plants (33.31 %) at 12 months after planting (12 MAP). In DAS-ELISA, lowest virus
titre with absorbance of 0.438 was observed in P. indica-colonized plants at 12 MAP,
compared to the highest virus titre in control plants (1.267). B. spectabilis treated
plants (0.596) also exhibited reduction in virus titre followed by P. fluorescens treated
plants (0.625) at 12 MAP. P. indica-colonized plants exhibited enhancement in growth
parameters viz., number of leaves (24.00), leaf area (1127 cm2), stem girth (42.74 cm)
and plant height (207.39 cm) at 12 MAP. P. indica-colonized plants also enhanced the
yield by 44.68 per cent followed by B. spectabilis treated plants (24.13 %) and P.
fluorescens treated plants (17.99 %). Moreover, fruits from P. indica-colonized plants
expressed significantly superior quality parameters.
Thus, findings from the present study could aid in the preliminary detection
and management of the virus, thus mitigating the widespread infection caused by
PRSV. The recombinant PRSV coat protein produced in this study could be used for
the development of PRSV antiserum. Additionally, our research highlights the
efficacy of eco-friendly management strategies for papaya ringspot disease, with
P. indica-colonization @ 106 cfu g-1 and also with four foliar sprayings as well as soil
drenching of B. spectabilis leaf extract (10 %) applied at fortnightly intervals, starting
from one month after planting. More field trials are to be conducted to integrate this
strategy in integrated disease management (IDM) package for the effective and
sustainable management of PRSD in papaya.