PhD Thesis

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    Etiology, molecular characterization and management of viral disease of dendrobium
    (Department of plant pathology , Vellayani, 2023-07-19) Safeer, M M.; Susha, S Thara
    The present study on “Etiology, molecular characterization, and management of virus diseases of Dendrobium” was conducted at the Department of Plant Pathology, College of Agriculture, Vellayani during the period 2015-2019. The study included survey among different orchid growers in Kerala, etiology of disease, detection of viruses using immunological and molecular techniques and finally finding a management strategy for the diseases. A scoring technique was developed for scoring severity of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) and based on which a survey was conducted among different orchid importers and growers in five districts of Kerala. Among a total of 9500 plants surveyed, a minimum five percent virus disease incidence was recorded in all locations surveyed. The present investigation observed the presence of CymMV and ORSV in the imported and cultivated Dendrobium orchids in Kerala. The study also observed that integrating meristem culture followed by root inoculation of P. indica in combination with foliar application of antiviral compounds effectively controls CymMV in Dendrobium orchids. This management practice can be easily adopted by farmers and with ease and had a prolonged effect on Dendrobium orchids.
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    Zinc homeostasis and carbon sequestration as influenced by biofertilizers and CO2 enrichment in rice (Oryza sativa L.)
    (Department of plant pathology, college of agriculture ,Vellayani, 2023-07-21) Amrutha, E A.; Manju, R V
    The study entitled "Zinc homeostasis and carbon sequestration as influenced by biofertilizers and CO2 enrichment in rice (Oryza sativa L.)" was undertaken with the objectives of assessing the impact of biofertilizers on Zn uptake, translocation and soil carbon sequestration and the mechanism of Zn transport as influenced by CO2 enrichment. The first experiment was conducted to study the influence of biofertilizers on Zn uptake, translocation, and carbon sequestration at IFSRS, Karamana. The experiment was laid out in RBD with eleven treatments and three replications. The treatments comprised of T1: PoP, KAU + Azolla, T2: PoP, KAU + AMF, T3: PoP, KAU + PGPR Mix I, T4: PoP, KAU + KSB, T5: PoP, KAU + PSB, T6: T1 + AMF, T7: T1 + PGPR Mix I, T8: T1 + KSB, T9: T1 + PSB, T10: Control and T11: PoP+ Zinc sulphate spray. The gene expression analysis revealed the down regulation of OsZIP4 genes under elevated CO2 compared to ambient CO2. Biofertilizer induced overexpression of OsZIP 4 genes is found to play significant role in improving Zn translocation. The study results could be helpful to formulate climate smart agricultural practices strategies to address the wide spread problem of limited availability of Zn among the population
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    Analysis of pathotypic variability of Xanthomonas oryzae pv.oryzae, the bacterial blight pathogen of rice and identification of new sources of resistance
    (Department of Plant Pathology, College of Agriculture, Vellanikkara, 2023-05-11) Aparna, V S.; Raji, P
    Bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is becoming a major production constraint worldwide. In India, the disease is causing considerable yield loss in all rice producing states. In Kerala, the disease is appearing in an epidemic form in recent years in Palakkad, Thrissur and Alappuzha districts. Host plant resistance offers a viable option for the management of the disease. Understanding the pathogen population prevailing in a geographical area is essential for the development of varieties having broad spectrum resistance. The present study was undertaken to analyse the pathotypic variability of Xoo in major rice growing areas of Kerala and to identify new sources of resistance.
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    Characterisation and management of sugarcane bacilliform virus (SCBV) causing leaf fleck disease in sugarcane
    (Department of Plant Pathology, College of Agriculture Vellanikkara, 2021) Sanju Balan; Anita Cherian
    Sugarcane (Saccharum officinarum) is a monocotyledonous perennial cash crop cultivated worldwide both under tropical and sub tropical conditions. It is being cultivated in more than 120 countries in the world. Like any other crops, it is also susceptible to biotic stress. Of which, diseases caused by viruses not only pose serious threat to sugarcane cultivation but also result in deterioration and exclusion of elite varieties of the germplasm. One of the major viral disease which affects global exchange of sugarcane germplasm is leaf fleck disease caused by Sugarcane bacilliform virus (SCBV). The research project entitled ‘Characterization and management of Sugarcane bacilliform virus causing leaf fleck in sugarcane’ was initiated with purposive sampling surveys in selected sugarcane fields in districts of Kerala and Tamil Nadu in order to document the symptoms under natural conditions, to assess the disease incidence, severity and to collect infected samples for further studies. The per cent disease incidence of the leaf fleck disease in Kerala ranged from 12 to 51 per cent whereas severity ranged from 10 to 36.5%. In Tamil Nadu the per cent disease incidence ranged from 28 to 56 per cent while severity ranged from 28 to 50.41%. Major symptoms observed on leaves were mottling, chlorotic flecks, chlorotic patches streaks and stripes with general yellowing of the canopy. In the case of severely affected clones, there was reduction in tillering, internodal length, number of internodes and appearance of deep longitudinal cracks. In highly susceptible clones, stunted growth with bunchy top appearance was noticed. On the basis of phenotypic variability of symptom expression, genotypes were classified into five groups. The development of the symptoms was also studied under artificial condition through insect transmission of the virus using pink mealy bug, Saccharicoccussacchari. Morphological characterisation of the virus done using electron microscopy revealed the presence of bacilliform virus particles of size 30 X 130–150 nm which indicated that the virus belongs to genus BADNA and family Caulimoviridae and the etiology of the disease was confirmed as Sugarcane bacilliform virus. The molecular detection of SCBV was also standardized through polymerase chain reaction (PCR). PCR amplification of RNaseH/RT gene was done using BADNA specific and SCBV129 specific primers. The amplicons were sequenced and in silco analysis of sequences showed sequence homology of 99 to 100 percent identity to SCBV. Widespread occurrence of the disease was observed even in the early generation of varietal development and in newly developed varieties. The transmission of the virus was suspected through true seed (fluff) developed by biparental crossing during sugarcane varietal development programme. Hence, the study was conducted to establish possible transmission of the virus from sugarcane parents to their progenies and the role of maternal and paternal parents in disease transmission through true seeds to the progenies. Samples from eight months old seedlings, three months old seedlings and parental clones were tested positive to the virus in PCR assays. Real time PCR was also standardized to assay these clones. Immunodiagnostic technique was validated using DAC ELISA. The technique of immunocapture PCR was also standardized. Minimal dilution of antisera with which SCBV could be detected was 2:1000 (V/V). Plant extract (antigen) at a dilution of 1:5 was found to be optimal for the detection of SCBV. Molecular detection of SCBV from mealy bug vector was also standardized. Both phenotypic and molecular methods were utilized to identify potential sources of natural resistance against SCBV. Based on the severity of symptom expression and PCR assays these were further classified as highly susceptible (HS), moderately susceptible (MS) moderately resistant (MR) and resistant (R). For generation of RNAi hair pin construct, initially forward (SF) and reverse primer (SR) were used to amplify 700 bp fragment of RT/RNase H gene to be cloned in sense orientation of the vector, pHANNIBAL. The linearized vector and the insert were ligated, and the ligation mixture was used to transform competent cells of Escherichia coli and the transformants were selected. Later antisense forward (AF) and reverse (AR) primer pairswere used to amplify 700 bp fragment of RT/RNase H gene to be cloned in antisense orientation. PCR product ligated into antisense direction of the vector and transformed into competent cells of E. coli. The recombinant pHANNIBAL vector was digested with restriction enzymes. The recombinant pHANNIBAL vector harbouringRNase H /RT gene was released from the vector through Not I site and subcloned into plant expression binary vector. Thus, cassette for RNA silencing was prepared.130 Meristem tip culture was also standardized with antiviral chemical tenofovir. Recovery percentage of meristem varied from 70 to 75 per cent and the viral load was quantified using real time PCR. The outcome of the study would facilitate early detection and elimination of the source of infection and prevent the spread of the disease in the field. Information generated in the study could be utilized while planning biparental crossing and reduce the spread of the virus in varietal development programmes. The hair pin construct developed in this study could be further utilized to generate transgenic disease resistant plants.
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    Development of acid tolerant strains of Bradyrhizobium sp. suitable for certain pulse crops of Kerala
    (Department of Plant Pathology, College of Agriculture, Vellayani, 1998) Meena Kumari, K S; Sasikumar Nair
    The present investigation on "Development of acid tolerant strains of Bradyrhizobium sp. suitable for certain pulse crops of Kerala" was conducted at College of Agriculture, Vellayani, Trivandrum during 1993-96. The initial isolation of acid tolerant strains of Bradyrhizobium suitable for cowpea, blackgram and green gram was done from seven different locations in Kerala. These locations were broadly grouped into category A and B. The root nodulation and plant dry weight were uniformly higher in category A locations compared to category B locations in all the three crops. In all, 43 native isolates of Bradyrhizobium were obtained including 17 from cowpea, 13 each from blackgram and greengram. The native isolates of Bradyrhizobium obtained were screened as per the Bureau of Indian Standard specification. The screening trial was done both under unamended and amended soil conditions. Thus in unamended soil, the isolates KA-G- C-4, VE-G-B-2 and PA-G-G-5 for cowpea, blackgram and green gram respectively were selected based on plant dry weight and in amended soil, the isolates KA-F-C-7, KA-F-B-6 and PI-G-G-8 for cowpea, blackgram and greengram respectively were selected for further studies. The colony characteristics of the selected isolates were typical of Bradyrhizobium forming white translucent glistening and elevated colonies with entire margin on YEMA with congored. All •the six isolates were gram negative and fast growers attaining satisfactory growth on YEMA within three days. The carbohydrate utilization pattern showed that arabinose, a pentose sugar was the most preferred carbon source for the native isolates. At the sametime, the growth of - the exotic isolates were maximum in glucose. The ability to tolerate low levels of acidic pH upto 4.5 was more for the native isolates. The growth of both these isolates gradually improved and attained a peak level at pH 7.5. The in vitro tolerance to Fe and Al were almost identical for both the native and exotic isolates. In general, the growth was progressively inhibited with an increase in the concentration of these elements from 50 to 250 ppm. The antibiotic resistant markers for ampicillin, streptomycin and kanamycin were same for both the native and exotic isolates. The growth of these isolates were not inhibited at concentrations upto 1000 ppm of ampicillin, 250 ppm of streptomycin and 50 ppm of kanamycin. In the serological characterisation it was observed that one of the heterologous antigens (PI-G-G-8) for green gram showed positive agglutination with that of the cowpea isolate, KA-F-C-7. The effect of application of FYM, liming or pelleting on root nodulation and plant growth characters in cowpea, blackgram and greengram was studied. Significant increases in nodule number, nodule dry weight and plant dry weight were obtained in the treatment combination of Bradyrhizobium inoculation along with FYM and lime application in all the three crops. An almost similar result was also obtained when liming was substituted with calcium carbonate pelleting. The efficacy of the selected isolates of cowpea, blackgram and green gram were tested under field conditions at two locations namely College of Agriculture, Vellayani and Rice Research Station, Kayamkulam during identical cropping season for two years. The seed treatment effects of the two selected Bradyrhizobium isolates for cowpea (KA-G-C-4 and KA-F-C-7), blackgram (VE-G-B-2 and KA-F-B-6), greengram (PA-G-G-5 and PI-G-G-8.) were evaluated with and without POP recommendations along with appropriate cont~ol treatments. In general, the root nodulation, plant growth and yield were significantly improved in all the three crops where Bradyrhizobium inoculation was practised along with the POP recommendation for each crop. Thus at Vellayani, the nodule number, nodule dry weight, leaf area, plant dry weight and yield in cowpea were maximum in the treatment combination of POP+KA-F-C-7. A similar response was also obtained with POP+KA- G-C-4 treatment. At Rice Research Station, Kayamkulam also, the yield increase was maximum in the treatment combinations of POP+KA- F-C-7 and POP+KA-G-C-4. In blackgram and greengram also the treatment effects were more or less similar to that of cowpea. In the last part of the present investigation, the influence of the POP recommendations on plant and soil nutrient status was studied. The nitrogen content of plants was significantly higher in . Bradyrhizobium inoculated plants in cowpea and blackgram at both the locations. In greengram, at Vellayani there were no significant differences between treatments in the NPK content of plants. At Rice Research Station, Kayamkulam, also significant increases in nitrogen content of plants were obtained due to Bradyrhizobium inoculation. Unlike the nitrogen content a uniform treatment effect was not observed in the P and K content of plants. In the studies on soil nutrient status, it was observed that in general the availability of P, K, Ca and Mg were increased in the treatment combinations where POP recommendation was practised along with Bradyrhizobium inoculation. However, there was no significant difference between treatments in soil pH, organic carbon, Fe and Al content of different soil samples.
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    Purification and serology of banana bunchy top virus
    (Department of Plant Pathology, College of Horticulture, Vellanikkara, 1998) Estelitta, S; Sukumara Varma, A
    Banana is one of the major fruit crop in Kerala and is often affected by the bunchytop disease caused by banana bunchytop virus. The disease is easily spread through infected suckers, which are used as the planting materials. Secondary spread is also seen through banana aphid, Pentalonia nigronervosa. Though field level quarantine measures may check the spread of the disease, rapid and convenient methods for the detection and identification of the virus in the suckers as well as in micropropagated plants have not been developed. In this background a study was designed and carried out to purify the BBTV, to produce antisera for developing a serological technique for the pre-symptomatic detection of virus in the planting materials of banana. Studies were also conducted to identify the type of nucleic acid of the virus and its morphology by direct electron microscopy. The study revealed that the disease incidence was maximum during August-November. The virus was not mechanically transmitted and tissue culture plants were the most susceptible planting materials for aphid transmission. Basic studies of virus-vector relationship were also conducted and the adult aphids were found to be effective vectors. In purification studies, among the different portions of banana plants used, the midribs of younger leaves yielded high concentration of the virus. Tissue culture plants yielded more virus concentration than other planting materials. Electron microscopy of the purified BBTV preparation revealed isometric particles of 18-22 nm size. Nucleic acids extracted from both healthy and infected samples were compared. The bands obtained were sensitive to DNase 1 and SI nuclease but not to RNase A, confirming the nucleic acid BBTV as ssDNA. SDS-PAGE analysis of BBTV coat protein revealed that it contained a major protein component of Mr 21000 with Rf value between that of β lactoglobulin (Mr 18400) and α chymotrypsinogen (Mr 25700). Antiserum of BBTV was produced in the rabbit and used for detection of virus specific antigens in different parts of the plant (midrib, petiole, leafsheath and rhizome) by chloroplast agglutination, agar gel diffusion, tube precipitation and ELISA. Among these methods ELISA was found to be highly sensitive for identification of the virus.
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    Interaction between VA mycorrhiza and bradyrhizobium in cowpea
    (Department of Plant Pathology, College of Horticulture, Vellanikkara, 1999) Beena, S; Rajendran Pillai, M V
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    Biochemical and biological bases of resistance in solanaceous vegetables against bacterial wilt incited by Ralstonia solanacearum (Smith) yabuuchi etal.
    (Department of Plant Pathology, College of Horticulture, Vellanikkara, 1998) Sheela Paul, T; James Mathew
    An investigation on biochemical, biological and nutritional bases of resistance in solanaceous vegetables against bacterial wi1t incited by Ralstonia solanacearum (Smith) Yabuuchi et al., has been attempted. The pathogen R. solanacearumwas isolated from respecti ve hosts and characterised by various morphological, cultural, biochemical and physiological tests upto biovar level. The three isolates were cross inoculable, and were sensitive to AmDistryn and Streptocycline. Based on these studies the tomato and chilli isolates were identified as Ralstonia solanacearum race I biovar III and that from brinjal as R. solanacearum race I biovar V. The study on toxigenic property of the bacterial isolates revealed that the toxic metabolites were not host specific. Out of the 43 varieties / lines screened , 12 varieties / lines were selected, one each from resistant, moderately resistant, moderately susceptible and susceptible categories. The varieties / lines selected were LE 79-5, BT-IO, LE 470 and Pusa Ruby for tomato; Swetha, Composite-2, BB-7 and Pusa Purple Long for brinjal and Ujwala, Manjari, Jwalasakhi and Pusa Jwala for chilli. The studies on biochemical, biological and nutritional factors in tomato, brinjal and chilli showed considerable variation between crops, between varieties/ lines, and between plant parts. However the root being the primary foci of infection by R. solanacearum the biochemical reactions in root is considered more important than other plant parts. Among the biochemical factors, the OD phenol and specific activity increased due to infection and the content was higher in the resistant genotype (LE 79-5) both under heal thy and diseased condition in tomato. In brinj al, the polyphenol oxidase activity, specific activity and peroxidase activity increased due to infection and were higher in resistant genotype (Swetha) both under healthy and diseased condition. In chilli, total phenol and OD phenol, increased due to infection and were higher in resistant plants (Ujwala) under healthy and diseased condi tions. The soluble sugar content and specific activity were also higher in resistant plants both under healthy and diseased condition even though a decrease was observed due to infection . In tomato, the resistant genotype showed a higher content of OD phenol, polyphenol oxidase activity, specific activity and alkaloids under healthy condition; and total phenol, OD phenol, soluble protein, specific activity and peroxidase activity under diseased condition. In brinjal, the resistant genotype recorded higher content of total phenol, OD phenol, soluble sugars, amino acids, soluble protein, polyphenol oxidase activity, specific activity and peroxidase activity under healthy condition; and polyphenol oxidase activity, specific activity and peroxidase activity under diseased condition. In chilli, the resistant genotype recorded higher total phenol, OD phenol, soluble sugars and specific acti vi ty under heal thy condition; and total phenol, OD phenol, soluble sugars, specific activity and peroxidase activity under diseased condition. Among the biological factors, the total microflora (fungi and actinomycetes), Pseudomonads and parasitic nematodes increased due to infection in resistant genotype whereas beneficial microbes recorded a decrease in population in resistant genotype by infection. In tomato the resistant genotype recorded higher nematode population under heal thy condi tion and higher total microflora, virulent Ralstonia and avirulent Pseudomonas under diseased condition. In brinjal the resistant genotype recorded higher population of fungi, avirulent Pseudomonas, mycorrhiza and saprophytic nematodes under heal thy condition, and fungi, virulent Ralstonia, avirulent Pseudomonas and nematodes under diseased condition. In chilli, the resistant genotype recorded higher populations of avirulent Pseudomonas, mycorrhiza and nematodes under heal thy condition and fungi, avirulent Pseudomonas and mycorrhiza under diseased condition. Among the nutritional factors, in tomato the resistant genotype recorded higher content of potassium and calcium under heal thy condition and iron, zinc and manganese under diseased condition. In brinjal the resistant genotype recorded higher content of nitrogen, calcium, magnesium and zinc under heal thy condition; and potassium, magnesium, iron and zinc under diseased condition. In chilli, the resistant genotype recorded higher content of phosphorus, calcium and iron under heal thy condition; and nitrogen, potassium magnesium and zinc under diseased condition. Thus the study revealed that it was not possible to arrive at common bases for resistance to bacterial wilt in tomato, brinjal and chilli taken together. However it was possible to outline the important parameters that conditions resistance in individual crops.
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    Strain variation in colletotrichum Gloeosporioides (Penz.) Penz & Sacc.
    (Department of Plant Pathology, College of Agriculture, Vellayani, 1996) Alice, K J; Karunakaran, P
    Colletotrichum gloeosporioides (Penz.) Penz. & SAcc. Is observed as a major pathogen infecting a number of crop plants, ornamentals and weeds. Detailed studies on the variability of this pathogen on important vegetables and ornamental plants in different locations of Thiruvananthapuram and Thirissur districts of Kerala were undertaken to distinguish different strains of C. gloeosporioides. Studies on the symptoms and pathogenecity tests of the 69 isolates showed that there were variations in the exoression of symptoms and in initiating symptoms indicating that the isolates were highly variable. Great variability in growth rate, colony characters, degree of sporulation and size of conidia were observed in different isolates. Based on the protein production and enzyme activity three groups of isolates could be distinguished. The isolates varied in their growth under varying temperature and relative humidity. Light intensity had no significant effect on the growth of the pathogen. Among the solid media tested, all the selected isolates exhibited the maximum growth on PDA. Richard’s medium was found to be the best liquid medium for mycelial growth. The degree of sporulation of selected isolates was the highest at 92.9 per cent RH and the lowest at 100 per cent RH. The highest sporulation was observed when exposed for 24 h of continuous light (125 lux) and poor when exposed for 24 h darkness. Basal medium was found to be best for sporulation. The results of host range studies and cross inoculation studies showed that the selected isolates were not host specific even though there were variations among the isolates in the intensity of infection. Inhibition of germination of selected seeds was observed when bioassayed with exotoxin. The purified toxin produced more or less identical symptoms as did by the casual organism. Coon’s medium was found to be the best for production of endotoxin. The maximum production of toxin was observed in 25 day old cultures. Biological groupings of all the 69 isolates studied were done based on the correlation matrix of different characters. Eight different groups of isolates could be distinguished and designated as G1, G2, G3, G4, G5, G6, G7 and G8 strains of C. gloeosporioides.