MSc.

Cowpea (Family: Fabaceae) is an important pulse cum vegetable crop of
suitable for the tropical and sub-tropical regions of the world. The grain type
cowpeas better tolerates the biotic and abiotic stresses against the vegetable types.
Under humid conditions, vegetable types, especially the pole types are susceptible
to many diseases and among them, anthracnose caused by Colletotrichum
lindemuthianum (Sacc. & Magn.) Br. and Cav. is very severe. In Kerala, complete
yield loss in vegetable cowpea is reported due to anthracnose during monsoons.
The study entitled “Characterization of pathogenesis related proteins for
anthracnose resistance in vegetable cowpea, Vigna spp.” was carried out with
objective to develop the protein profiles of resistant and susceptible bush and pole
genotypes through SDS-PAGE analysis at different time intervals of infection and
to characterize the differentially expressed proteins by MALDI-TOF followed by
in-silico analyses.
Two bush type varieties Pusa Komal and Kanakamony, the former
reported to be highly susceptible and the latter immune to anthracnose and two
pole type varieties Lola and Arimbra Local, of which the former susceptible and
the latter resistant were used in the study. Pure culture of the pathogenic fungus
was developed and maintained on selective medium (Neopeptone-Glucose-Agar)
at the Dept. of Plant Pathology. The identity of Colletotrichum lindemuthianum
has been established from the spore characteristics observed under phase contrast
microscope and the pathogenicity was confirmed through artificial inoculation
under controlled conditions. The pot culture experiment was conducted with 50
pots per variety. Artificial inoculation of pathogenic fungus was done and the leaf
samples were collected at 0, 6, 12, 18, 24, 48, 72, 96, 120, 144,168 and 192 hours
after artificial inoculation. The total protein was extracted using Tris-HCl buffer
(pH-7.5), quantified using spectrophotometer and analyzed by SDS-PAGE
method. The defense enzymes like peroxidase (PO), polyphenol oxidase (PPO)
and phenylalanine ammonia-lyase (PAL) were assayed.
By artificial inoculation, disease responses for anthracnose were confirmed
to be highly susceptible in Pusa Komal and Lola; highly resistant in Arimbra
Local and immune in Kanakamony. Protein expression was found to be higher
from the initial hours in resistant varieties whereas in susceptible varieties, the
expression was reduced immediately after infection then peaked at 18hr and
gradually decreased later on. Two prominent and differentially expressed protein
bands at 56 kD and 14 kD were sequenced in MALDI-TOF to obtain the peptide
mass fingerprint. Through in-silico analyses using Mascot server software, they
were identified to be the large and small subunits of the chloroplastic enzyme
RuBisCo. Thus the capability of a variety to maintain high levels of RuBisCo was
found to be the deciding factor for anthracnose disease resistance. Further, protein
profiles developed after purification of proteins by dialysis have clearly identified
the differentially expressed band at 29 kD in the resistant varieties which is in the
size range of already reported PR proteins.
PO and PAL activities were proportionate to the resistance behavior, with
the peak values at 18 and 24 hr after inoculation. With the results of this study,
these defense enzymes are recommended as biochemical markers for identifying
the resistance in the accessions. Capability to maintain higher levels of RuBisCo,
PO and PAL enzymes is the characteristic of anthracnose resistant vegetable
cowpeas and the future breeding programmes could be oriented in this direction