PhD Thesis
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Item Plant growth promotion and stress mitigation mediated by the root endophytic fungus Piriformospora indica In Cassava (Manihot esculenta Crantz)(Department of Microbiology, College of Agriculture, Vellayani, 2025-12-12) Akhila P Subhash; Anith, K NThe study entitled “Plant growth promotion and stress mitigation mediated by the root endophytic fungus Piriformospora indica in cassava (Manihot esculenta Crantz)” was conducted in the Department of Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram and in the ICAR–Central Tuber Crops Research Institute (CTCRI), Sreekariyam, Thiruvananthapuram during 2021-2024, with an objective to investigate the effects of the root endophytic fungus P. indica in cassava on productivity, nutrient acquisition and disease suppression against major pathogens. Other microbial agents used in the study were arbuscular mycorrhizal fungus Glomus mosseae, and the biocontrol bacterium Pseudomonas fluorescens PN026. Initially, the pathogens causing stem and root rot disease in cassava were isolated from the naturally infected plants. Three fungal isolates were obtained from the infected cassava samples, and these isolates were identified as Lasiodiplodia theobromae, Fusarium sp. and Cunninghamella elegans with morphological studies and by molecular identification using internal transcribed spacer region (ITS) sequence-based technique. Among them, L. theobromae and Fusarium sp. were identified as the major pathogens and C. elegans as a minor pathogen causing the disease. Pathogenicity of the isolates was verified by proving Koch’s postulates by inoculating the isolated pathogens separately, sequentially and in different combinations in freshly harvested healthy cassava tubers. Treatment with the combination of pathogens L. theobromae and Fusarium sp. was identified as the one showing highest infectivity with high percentage of weight loss and with least number of days taken for the symptom development. This inoculum combination was used for further studies. Direct and indirect antagonism of P. indica against the cassava stem and root rot fungal pathogens was evaluated on potato dextrose agar (PDA) plates. Direct antagonism was evaluated through dual culture plate assay and indirect antagonism through agar well diffusion method using the culture filtrate of P. indica. However, in both the tests P. indica did not inhibit these fungal pathogens in vitro. In vivo studies included cassava root colonization and root initiation, nutrient acquisition and plant growth promotion experiment as well as stem and root rot disease and cassava mosaic disease suppression. An experiment was conducted in protray to evaluate the root colonization abilities of P. indica in Sree Suvarna, Sree Jaya and Aswathy varieties of cassava in both sterile and unsterile planting medium. Fungal structures were detected by trypan blue staining, WGA-AF 488 (Wheat germ agglutinin- Alexa flour 488) staining and confocal imaging. Pear shaped chlamydospores were observed within the root cortical tissues at 14 days after inoculation. Further confirmation was done by molecular detection with the help PiTEF gene primer using a PCR reaction. This is the first report of successful colonization of P. indica in cassava. The root colonization ability of the AM fungus G. mosseae in all the three varieties of cassava in sterile planting medium was also evaluated using trypan blue staining method. The percentage of root colonization was 100 percent in all these varieties, where AM fungal vesicles as well as arbuscles were observed in the roots at 14 days after inoculation. Root initiation tests in minisetts were carried out in Sree Jaya and Aswathy cassava varieties by applying P. indica and Pseudomonas fluorescens in both sterile and unsterile planting medium. When P. indica and P. fluorescens colonized plants were compared to control plants, all root growth parameters were observed to be higher. However, most of parameters were found significant in unsterile planting medium when compared to the sterile planting medium which may be due to the insufficient root colonization of both the beneficial microbes in cassava minisetts in the sterile medium. A field experiment was conducted by applying P. indica and AM fungus G. mosseae, along with different doses of NPK fertilizers in cassava variety Sree Suvarna to evaluate the abilities of both beneficial fungi to promote growth and acquire nutrients. Different biometric, physiological, yield as well as quality parameters were analyzed. The application with a combination of 75 percent of the recommended dose of NPK along with P. indica was found to be the best treatment that improved growth and yield attributes, whereas the combination of 75 percent NPK along with G. mosseae recorded significant improvement in total dry matter contents as well as tuber quality parameters. The results showed that the chemical fertilizer dose could be reduced to a level of 75 percent of the recommended dose, without compromising the growth and yield, if the fertilizers are applied together with the bio-inoculants, either P. indica or G. mosseae. The same treatments also resulted in higher total nitrogen uptake while the combination of 100 percent NPK and G. mosseae resulted in better phosphorous and potassium uptake by the cassava plants. A grow bag experiment was conducted to examine the stem and root rot disease suppressing ability of P. indica in the local disease-prone cassava variety Aswathy. The treatment combination of pathogens, L. theobromae and Fusarium sp. was selected for the inoculation purpose. An increase in the growth and yield and decrease in disease incidence was noted in P. indica and G. mosseae inoculated plants, even when the plants were challenged with the stem and root rot pathogens. The pathogen inoculated control plants recorded the least number of days taken for the initial symptom development (6 months after planting), highest percentage of tubers affected per plant (53.33%) and extent of damage in each tuber (50%) followed by the chemical control plants. Simultaneously another grow bag experiment was also conducted, in the mosaic susceptible variety Sree Jaya to assess the cassava mosaic virus disease suppression by P. indica. Natural incidence of cassava mosaic virus in the cassava plants was monitored by visual scoring from 4 MAP till harvest (7 MAP). The plants from absolute control recorded highest visible symptoms of mosaic disease which was on par with G. mosseae treated plants, and least symptoms were recorded in the P. indica treated plants. Viral load in the cassava leaves estimated by quantitative polymerase chain reaction (qPCR) showed highest viral copy number in the G. mosseae treated plants and least in the P. indica treated plants. However, the G. mosseae treated plants showed maximum yield parameters compared to all other treatments. From the results of the present study, it can be inferred that the combined application of P. indica (vermiculite based inoculum one percent w/v @ 20 g/pit) or AM fungus G. mosseae (@ 5 g/pit) along with 75 percent recommended dosage of NPK fertilizers at the time of planting helps to reduce the amount of chemical fertilizers as well as enhance the growth and yield parameters of cassava plants. The study also suggests that P. indica as well as AM fungus G. mosseae can be utilized as biocontrol agents to combat stem and root rot disease of cassava. Effective colonization of P. indica also reduces the incidence of cassava mosaic virus. The combined application of these two beneficial endophytic fungi might have the potential to improve the growth and yield parameters of cassava with positive effects on disease control.Item Nutrient management in cassava (Manihot esculenta Crantz) variety Vellayani Hraswa for southern laterites (Agro ecological unit 8)(Department of Agronomy, College of Agriculture ,Vellayani, 2024-06-01) Aparna, R A.Phosphorus transformation in acid sulphate rice soils of Kerala Acid sulphate soils are lowland soils situated below the mean sea level along the coastal tracts of Kottayam, Alappuzha, Thrissur and Malappuram districts of Kerala, spread in two agro-ecological units namely Kuttanad (AEU 4) and Kole lands (AEU 6). These soils are characterised by high levels of organic matter, low pH and toxicity of iron (Fe) and aluminium (Al) with wide variability in available phosphorus (P) content. Acid sulphate soils generally contain substantial quantities of Fe sulphide minerals or the oxidation reaction products of these sulphidic minerals and the protonated/non-protonated surfaces of Al/Fe hydroxides and oxides, resulting in P sorption and formation of the complexes, thus decreasing P availability in soil. However, the recent fertility evaluations across the state showed high available P status due to solubilization of the accumulated applied P. To develop an economically viable phosphorus nutrient management strategy, a pioneer study on P transformations in acid sulphate soils is highly required. The study aimed to understand seasonal variation of nutrients and transformations of phosphorus in acid sulphate soils. The investigation was conducted at Radiotracer laboratory, College of Agriculture, Vellanikkara, with four experiments viz., collection and characterization of soil samples from the acid sulphate soils of Kerala in two seasons, fractionation of phosphorus, adsorption study and an incubation study. In the first experiment, 125 representative soil samples from Kuttanad (AEU 4) (15 each from Upper Kuttanad, Lower Kuttanad, Vechur Kari, Purakkad Kari and Kayal lands) and Kole lands (25 each from Thrissur and Ponnani Kole) in pre-monsoon (April) and post-monsoon (November) were collected and characterised. The experiments on the fractionation and adsorption of phosphorus were done in thirty-five samples ie., five each from the seven selected locations with varying available P status. Phosphorus fractionation was carried out to quantify different fractions of phosphorus and their contribution to the available pool. The inorganic P fractions includes saloid bound P, Fe-P, Al-P, reductant soluble P and Ca-P. Adsorption study was carried out to work out the quantity/ intensity (Q/I) relationship of phosphorus and the pattern of adsorption in acid sulphate soils. Finally, an incubation experiment of 90 days duration was conducted to understand the effect of phosphatic fertilisers, lime and farmyard manure at different levels in low and high available P soils collected from Kuttanad and Kole lands. The treatment details of the incubation study were three levels of phosphatic fertilisers (as SSP - 0, 35 and 70 kg ha-1), two levels of lime (as CaO - without lime and lime as per POP recommendations) and two levels of farmyard manure (0 t ha-1 and 5 t ha-1). The observations were made on 30th, 60th and 90th days of incubation. Seasonal characterisation of soil samples during the two seasons revealed that among the collected samples, 50 per cent was sandy clay loam and 40 per cent was sandy loam in texture. The acidity characterisation of soil samples reaffirmed the extreme acidity condition of acid sulphate soils with 40 per cent of samples coming under extremely acidic class and 20 per cent under very strongly acidic. Among the collected soil samples, 37.60 per cent was low in available P, 23.20 per cent and 39.20 per cent under medium and high P respectively. The soil sample from Vechur Kari region showed the highest value of organic carbon (11.46 %). The available micronutrients viz., Fe and Mn showed very high concentrations. Among the acidity fractions, potential acidity was dominant followed by pH-dependent acidity in both seasons. Seasonal variation in electrochemical properties like pH and EC was noticed between two seasons. Ultra acidic soil reaction with high electrical conductivity was noticed during pre-monsoon period which reaffirm the salt water intrusion in these locations. Oxidation of pyrite mineral resulted in high amount of available sulphur as well as potential acidity during pre-monsoon. Reduced condition prevailing in the post monsoon resulted in high concentration of available iron. The fractionation study of phosphorus indicated that the per cent distribution of different phosphorus fractions followed the order, Fe- P > organic P > reductant soluble P > calcium P > aluminium P > saloid bound P. The contribution of dominant fraction (Fe-P) to the available P is mainly through saloid bound P. The phosphorus fixing capacity was significantly and positively correlated with clay, organic matter content, and negatively correlated with the available P. In the adsorption study, high buffer power indicated the ability of acid sulphate soils to replenish the depleted available P. Freundlich adsorption isotherm was found to be the best to explain P adsorption followed by Langmuir and Temkin adsorption isotherms. The Freundlich adsorption constant, KF was found to be correlated with organic matter which confirmed the fixation of phosphorus by organic matter in acid sulphate soils. In most of the soils, Langmuir adsorption constant (KL) increased with rise in temperature, indicates chemisorption behaviour of P adsorption. The incubation study affirmed that in soils with low available P, the addition of SSP at doses of 70 kg ha-1 and 35 kg ha-1 along with lime and FYM showed a significant increase in pH. In these soils, the addition of SSP (70 kg ha-1) along with lime and FYM registered the highest available P of 51.63 and 73.33 kg ha-1 respectively in Kuttanad and Kole soils at 60th day of incubation. In the same treatment, Fe bound P fraction was found to be highest with a decreasing trend towards 60th day of incubation, which increased after that. In the case of soils with high available P, the treatment combination with the addition of the highest dose of SSP (70 kg ha-1) along with lime and FYM showed the highest pH with an increasing trend towards 60th day. On the contrary to low P soils, in high P soils, the addition of SSP at 35 kg ha-1 along with lime and FYM registered the highest available P of 309.44 and 126.94 kg ha-1 respectively in Kuttanad and Kole at 30th day of incubation followed by a reduction towards 60th day of incubation. Phosphorus, a widely varying nutrient in acid sulphate soils is prone to high adsorption and fixation in soil depending upon the type of clay, organic matter content and amount of oxides and hydroxides of Fe and Al. There was no significant seasonal variation of P in soil samples from AEU 4 and AEU 6. In acid sulphate soils, the most dominant fraction, Fe-P contributed to available P mainly through saloid bound P, which is the water-soluble and loosely bound P fraction. The reduction of available P with the increase in P fixing capacity necessitates its estimation at least for the grouping of soils for the efficient management of phosphorus. In soils with high available P, addition of lower dose of SSP (35 kg ha-1) along with organic manure (FYM @ 5 t ha- 1) and lime (POP recommendations) is sufficient to enhance the P availability. Where as, in soils with low available P, addition of higher dose of SSP (70 kg ha-1) along with organic manure (FYM @ 5 t ha-1) and lime (POP recommendations) enhanced the P availability than its sole application. In this regard, field experiments in acid sulphate soils have to be conducted to confirm the results of incubation study in rice under natural system and more investigation is needed to know the interaction between organic matter and organic P fraction. As the organic matter plays the dual function in the environment- as a link and as a bottleneck for phosphorus availability, detailed study should be undertaken to know the complex formed by P fixation and its degree of crystallinity for developing better P management strategies in acid sulphate soils.Item Identification of graft transmissible resistant factors and development of si RNA mediated resistance in cassava against cassava mosaic geminivirus(Department of Plant Pathology, College of Agriculture, Vellayani, 2017) Asha B Nair; Umamaheswaran, KItem Introgression of mosaic resistance in popular short duration cassava varieties of Kerala through marker assisted selection(Deparment of Plant Breeding and Genetics, College of Agriculture, Vellayani, 2018) Darshan, S; Arya, KThe present study entitled “Introgression of mosaic disease resistance in to popular short duration cassava varieties of Kerala through marker assisted breeding”was conducted in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, Kerala Agricultural University and Division of Crop Improvement, ICAR- Central Tuber Crops research Institute, Sreekariyam, Thiruvananthapuram, Kerala during the period 2014 - 2017 with the core objective of introgression of cassava mosaic disease (CMD) resistance to short duration varieties of cassava through marker assisted selection (MAS) and to study the inheritance of early bulking nature. The research work was carried out as four experiments. In the first experiment, Five early bulking high yielding lines viz, Sree Jaya, Sree Vijaya, Vellayani Hraswa, CI 889 and 9S 75 and three testers viz, CR 54A3, IMS2-5 and CI 273 with resistance to cassava mosaic disease were selected and planted in a pollination block and crossed in Line x Tester (LxT) design to produce hybrid seeds of 15 F1 combinations. Experiment II was conducted in two parts. Screening of F1 seedlings for CMD resistance and early bulking nature was carried out in the first part of experiment II, where hybrids along with the parents were evaluated. Analysis of variance revealed significant differences among the genotypes for all the traits studied. All the agronomic traits were recorded and inheritance of early bulking and its correlation with other traits were studied. The CMD incidence expressed significant and negative correlation with tuber yield per plant where as significant and positive correlation for all other traits with tuber yield per plant was observed among the F1’s. As a part of experiment II (b), seedlings without the CMD visual symptoms were subjected to multiplex PCR and the results revealed that among the parents Sree Jaya, Sree Vijaya, Vellayani Hraswa expressed presence of Srilankan Cassava mosaic Virus (SLCMV) and Vellayani Hraswa expressed the presence of both SLCMV and Indian Cassava mosaic Virus (ICMV). Among the crosses, Sree Jaya x CR54 A3 (L1x T1), Sree Jaya x IMS2-5 (L1xT2), Vellayani Hraswa x IMS2-5 (L3xT2), CI 889 x CI 273 (L4xT3) expressed the presence of SLCMV. Real time PCR (qPCR) assay for seedlings identified CI 889 (L4), 9S 75(L5), CR 54A3 (T1), IMS2-5 (T2) and CI 273(T3) among the parents and Sree Jaya x CR54 A3 (L1x T1), Sree Jaya x IMS2-5 (L1xT2), Sree Jaya x CI 273(L1x T3) and 9S 75 x CR54 A3 (L5x T1), 9S 75 x IMS2-5 (L5xT2) and 9S 75 x CI 273 (L5x T3) among the crosses as highly resistant, based on viral load present in the DNA sample. Based on the previous report ten CMD resistance linked markers were screened through BSA and five of which SSRY 28, SSRY 44 SSRY 40, SSRY 106 and SSRY 235 were selected. Among the CMD linked SSR markers studied, the maximum polymorphism was elucidated by SSRY 28, SSRY 44 and followed by SSRY 235. SSRY 28 is a strongly linked marker to CMD2 which is a dominant gene conferring resistance among the clones of combinations (L1xT1, L2xT2, L3xT1 and L3xT3) three of five markers revealed alleles associated with CMD2 gene In the third experiment to evaluate the early bulking clones, field was laid out in randomized block design (RBD) with three replications consisting of CMD resistant clones along with parental clones using miniset technique. Analysis of variance revealed significant differences among the genotypes for all the traits. Measurement of heterosis was carried out considering parent Vellayani Hraswa (L3) as check and results revealed that standard heterosis was positive and significant in the combinations Sree Jaya x CR 54A3 (L1xT1) and Sree Jaya x CI 273 (L1xT3) for all the yield contributing traits. The crosses Sree Jaya x CR54 A3 (L1x T1) and Sree Jaya x CI 273 (L1xT3) exhibited negative standard heterosis for CMD. Combining ability analysis showed significant gca, sca variances and gca, sca effects for all the traits. Moreover gca/sca variance ratio indicated preponderance of dominance / non-additive gene action for the inheritance of all traits. Among the lines, Sree Jaya (L1) exhibited positive and significant gcaeffect for tuber yield and yield contributing traits. Among the testers, IMS2-5 (T2) exhibited negative and significant gca effect for CMD. Among the crosses Sree Jaya x CR54 A3 (L1x T1) exhibited positive and significant scaeffect for girth of tuber and stem girth, 9S 75 x CI 273 (L5xT3) exhibited positive and significant scaeffect for tuber yield per plant, CI 889 x CR 54A3 (L4xT1) exhibited negative and significant scaeffect for CMD. In the last experiment, through bulk segregants analysis using 5 SSR markers linked to early bulking in cassava were selected out of 9 SSR markers selected. Among 5 SSR markers of CMD and early bulking nature two SSR markers (SSRY 28 and SSRY 106) associated with resistance to CMD and One SSR marker, ESTs (SSRY) 292 associated to early bulking nature has been identified. Among the crosses, clones from Sree Jaya x CR54 A3 (L1xT1), Sree Jaya x CI 273 (L1x T3) and 9S 75 x CR 54A3 (L5xT1) are being confirmed with CMD resistance as well as early bulking nature.