PhD

A study entitled “Nutrio-physiological and molecular analyses and carbon
dioxide enrichment studies of coconut palms (Cocos nucifera L.) with foliar
yellowing” was carried out with
the objective to analyze the nutrio -
physiological changes occurring in the palms affected with foliar yellowing and to
assess the impact of enhanced carbon dioxide on phytoplasmal response. In this
study, palms showing mid whorl yellowing (MWY), root (wilt) affected palms
(RW) and healthy palms were selected from two different locations viz the
Instructional Farm, College of Agriculture, Vellayani (location 1) and Venganoor
region (location 2) in the Thiruvananthapuram district. Coconut seedlings
showing yellowing were selected from the Instructional Farm, College of
Agriculture, Vellayani to study the effect of enhanced carbon dioxide on
phytoplasmal response. Catharanthus and brinjal plants with ‘little leaf symptom’
maintained at the Department of Plant Pathology were utilised for grafting into
healthy catharanthus and brinjal plants and these were also taken for studying the
phytoplasmal response under elevated carbon dioxide condition.
Physiological and biochemical analyses revealed significant variations in
all the parameters studied. Palms with mid whorl yellowing (MWY) showed a
significant reduction in the chlorophyll a, b and total chlorophyll contents, relative
water content, membrane integrity and phenol content compared to the healthy
palms. An increase in the protein (88%), carbohydrate (25.14%), reducing sugar
(28%) and starch content (28.33%) was noted in palms with MWY. In the case of
antioxidant enzymes there was build up of polyphenol oxidase (145.38%) and
peroxidase activities in palms with MWY compared to the healthy palms. A
similar trend was observed in almost all cases of RW affected palms.
Nutrient analyses revealed significant alterations in the nutrient content of
the selected palms. Significant reduction in the nitrogen (47.74%), magnesium
(22.72%) and zinc content (24.19%) was observed in palms with yellowing
compared to the healthy palms. Accumulation of elements like potassium
(37.96%) calcium (40.79%), iron (54%) and copper (35.75%) was observed in
palms with mid whorl yellowing compared to the healthy palms. These variations
in the nutrient levels can have an influence on symptom development in coconut
palms. The results on the soil nutrient analyses did not show any particular role in
development of yellowing symptoms.
Phytoplasma cells were detected in the phloem tissues of both mid whorl
yellowing and root wilt affected palms under Scanning Electron Microscopy
(SEM) study. Morphological and anatomical analyses of fresh roots indicated
browning and necrosis of roots and vascular disintegration in MWY and RW
affected palms.
The presence of phytoplasma in the selected samples were checked by
nested PCR analysis using phytoplasma specific universal primers-P1/P7-
R16F2n/ R16R2, Phytoplasma 16S rDNA specific semi nested primers 1F7/7R3 -
1F7/7R2 and by real time PCR technique using real time primers QPF2/R2. Semi-
nested PCR yielded an amplicon of 493 bp in all the MWY and RW palms in both
the locations. Real time PCR yielded an amplicon of 140 bp in the RW palms, 3
MWY palms in location 1. Nested PCR with phytoplasma specific universal
primer pairs P1/P7-R16F2n/ R16R2 yielded an amplicon of 1.2 kb in MWY palm
and RW palm. Sequence analysis of the mid whorl yellowing phytoplasma
revealed 89% similarity to the root wilt phytoplasma.
In experiment II, nested PCR with universal primer pairs showed no
amplification in the coconut seedlings and hence only catharanthus and brinjal
grafts with phytoplasmal infection were kept in Open Top Chamber (OTC) with
500 ppm Carbon dioxide concentration for a period of one month. After the
exposure period, the plants kept in OTC showed an increased rate of growth and
development with phytoplasmal symptoms. Physiological and biochemical
analyses showed a significant increase in the reducing sugar content (63%),
protein content (147%) and PAL activity (32%) and significant reduction in the
phenol content (37%), and polyphenol oxidase activity (62%) in the infected
plants kept in OTC compared to the infected plants kept in open condition.
Molecular analyses of the infected samples using real time PCR kept in OTC and
open condition with the DNA samples isolated at the 15th and 30th day of exposure
was done. No significant variation was observed in the phytoplasmal load after
elevated carbon dioxide exposure.
The role of phytoplasma in causing MWY was established by molecular
and anatomical studies. Sequencing of the PCR product revealed that MWY
phytoplasma can be a variant of root (wilt) phytoplasma. The present study
indicated that the overall health status of the palms with MWY was highly
deteriorated. Significant variation in the nutrient profile was noted which
emphasise the need for proper nutrient management. Semi- nested PCR was found
to be more accurate and specific in phytoplasmal detection which can be utilised
for phytoplasmal indexing and mother palm and disease free seedling selection.
Elevated carbon dioxide showed an improved growth and development and
activated defense response to phytoplasma in the infected plants but the CO2
induced modifications in phytoplasmal load was not detected. The results
indicated a better tolerance strategy to
environmental conditions.
phytoplasma under
changing