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2011 - 20172
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Author: Deepa, S × End date: 2017 × Start date: 2010 ×
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    ThesisItemOpen Access
    Nutrio-physiological and molecular analyses and carbon dioxide enrichment studies of coconut palms (Cocos nucifera L.) with foliar yellowing
    (Department of Plant Physiology, College of Agriculture, Vellayani, 2017) Deepa, S; KAU; Manju, R V
    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
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    ThesisItemOpen Access
    Physiological, anatomical and molecular analysis of coconut palms (Cocos nucifera L.) affected with yellowing.
    (Department of Plant Physiology,College of Agriculture, Vellayani, 2011) Deepa, S; KAU; Manju, R V
    Kerala the ‘Land of coconut’ occupies first position in area and production of coconut in India but the productivity is below the national average. This is because of unproductive and senile palms, lack of adaptation of recommended cultivation practises and serious incidence of pests and diseases. The root (wilt), an endemic disease of Kerala causes an annual loss of 968 million nuts. Recently another type of yellowing characterised by mid whorl yellowing shedding of immature nuts and drying of inflorescence without showing characteristic ribbing symptom is rapidly spreading in many parts of Kerala. There is no research report available in this emerging problem in the coconut growing tracts of Kerala. Since there is rampant spreading of yellowing to other coconut cultivated areas causing a serious reduction in nut yield, this problem demands immediate attention. Hence an experiment was conducted in the Department of Plant Physiology, College of Agriculture, Vellyani to generate the basic information on the causative agents and on the changes of physiological, molecular and anatomical realms of coconut affected by mid whorl yellowing. A wide variation was observed in the physiological parameters like total pigments, relative water content, carbohydrate fractions, proteins, phenols and antioxidants betweenpalms with yellowing and control palms. The palms with yellowing showed a significant reduction in the total chlorophyll, chlorophyll a, chlorophyll b and carotenoid content when compared to the healthy palm. Relative water content was lower for all the palms with yellowing compared to the control palm and the healthy palms were maintaining a better water status and membrane integrity. All the selected palms with yellowing recorded an accumulation of reducing sugars compared to the healthy palms. The trend was similar for starch content except for palms with general yellowing. The control palms recorded maximum protein content compared to other palms with yellowing. A significant increase in the phenolic compounds was observed in palms with yellowing compared to the healthy palms. This results point to an activated defense system. The 14th leaf starting from the first fully opened one is the most widely used leaf for nutrient analysis as recommended by the IRHO. (Fremond et al., 1966).The 14th leaf data from the control palms and palms with different types of yellowing were analysed. The data showed that there were significant reductions in the levels of major nutrient nitrogen, phosphorus and also in the levels of magnesium and the micronutrient manganese in case of palms showing mid whorl yellowing compared to the control palm. But significantly higher levels of accumulation were found in case of potassium, calcium, iron and copper. These variations in the nutrient levels can have an influence on symptom development in coconut palms. The maximum yield was recorded for the control palm. Chlorophyll degradation and loss of structural integrity were evident from the anatomical studies of leaf tissues in case of palms with yellowing. Vascular browning which extended to the cortex and the vascular disintegration in the roots of affected palms were seen on anatomical analyses. A pure quality DNA was obtained by modified CTAB DNA isolation protocol. Molecular analyses using nested PCR showed that with the phytoplasma specific universal primers P1/P7-R16F2n/R16R2 provided an amplicon ) in the positive control and palms with inner and middle whorl yellowing and the primers R16mF2 /R16mR1 -R16F2n/ R16R2 provided an amplicon for the positive control and palms with inner, middle and general yellowing. The root (wilt) phytoplasma specific semi- nested primers IF7/7R3- IF7/7R2 provided an amplicon of 493 bp only for the positive control and no amplification was seen in any of the selected palms. The control palm and the palms affected with outer whorl yellowing provided no amplification at all for any of the primers tested. Amplification of phytoplasma specific universal primers in palms with mid whorl yellowing indicates that phytoplasma has got a role in development of the specific symptom. This demands immediate attention and extensive studies on the etiology, changes in biochemical profile, anatomy etc. Salient Findings Amplification of phytoplasma specific universal primers in palms with mid whorl yellowing indicates that phytoplasma has got a role in development of the mid whorl yellowing symptom. But in case of the root (wilt) phytoplasma specific primers, there was no amplification in palms with mid whorl yellowing. This suggests that the mid whorl yellowing of coconut palm which is an emerging problem in Kerala may not be associated with the root (wilt) disease. The results of biochemical and physiological analyses of the selected palms clearly indicate an altered primary metabolism, source-sink relation and a complexity in the nutriophysiology. These altered conditions can act as predisposing factors for the development of specific symptoms. The anatomical analyses showed damages of roots in the palms with mid whorl yellowing. They will have internal browning of vascular elements, extending into the cortex and also disintegration of vascular elements. Future line of work Detailed analysis of nutriophysiology, source-sink relation and hormonal profile. Studies on soil factors like nutrient level and water status. Extension of anatomical studies to different type of tissues. Extension of molecular studies into identification of specific phytoplasmal strains associated with mid whorl yellowing of coconut palms. Development of management strategy for the improvement of the field performance and productivity of coconut palms with mid whorl yellowing.