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 Evaluation and characterisation of selected landraces of rice for bioactive compounds and validation of its nutraceutical potential through in silico analysis(Department of Plant Physiology, College of Agriculture ,Vellanikkara, 2024-05-21) Mikhina,M S.; Abida, P SEvaluation and characterization of selected landraces of rice for bioactive compounds and validation of its nutraceutical potential through in silico analysis Abstract Nearly ninety per cent of the world's production of rice comes from Asian nations, where it is an often-cultivated crop. The world produced 513.68 million tonnes of rice in 2022–2023, with China producing the most at 149 million tonnes, followed by India with 132 million tonnes. Rice is consumed by nearly one-third of the global population accounting for about 80 per cent of their energy requirements. In addition to being the staple grain of Asia, rice is quickly taking the lead as the main diet in Latin America and Africa. Oryza sativa is the most widely grown species of the available approximately 11,000 rice types and 100,000 landraces as well as improved cultivars. Existence of such a huge genetic diversity enables humans to improve the crop’s performance in spite of changing climatic scenarios and burgeoning population. Traditional rices are enriched with amino acids, antioxidants, phytochemicals, vitamin E and other nutrients. In addition to their antioxidant activity, traditional rice varieties offer anti-diabetic and anti-inflammatory benefits. Epidemiological studies have shown that regular consumption of whole grain is associated with reduced risks of chronic diseases such as cardiovascular diseases, type 2 diabetes and some cancers. Ayurveda considers traditional whole rice grains as nourishing food and medicine. Brown rice and red rice are great sources of fiber, B vitamins, calcium, zinc, iron, manganese, selenium, magnesium and other nutrients. Due to its higher digestibility, nutritional quality, potential health benefits and biological activity, rice is categorized as the queen of cereals. But these rices are underexploited and requires a boost in yielding potential, for their commercialization. In this background, the present study was envisaged to delve into the nutraceutical superiority of a few selected speciality rice and highlight the significance of conserving and protecting them. The study was undertaken as four experiments spread across College of Agriculture, Vellanikkara, RARS, Pattambi and CFTRI, Mysore. As first part of the study, a pot culture experiment was conducted for morpho-physio and biochemical evaluation of the selected twenty-five rice landraces, laid in completely randomised design. Among the selected landraces, Kavunginpoothala recorded significantly highest plant height (109.56 cm), followed by Chenkayama (101.51 cm), while Ithikandapan was the shortest (93.93 cm). The local check Jyothi and Uma showed a plant height of 96.35 cm and 103.35 cm respectively. Maximum number of tillers were observed in Palthondi (6) which was on par with Chenkayama, Chuvannavattan, Thavalakkannan, Rakthasali and Japan Violet. Black jasmine (4) possessed the least number of tillers and was on par with Kala namak and Krishnakaumudh. Among the selected landraces, the number of panicles of Palthondi (6) Chenkayama, Thavalakkannan, Rakthasali, Japan Violet and Chuvanna Vattan were on par with each other. The local check Jyothi possessed significantly higher number of panicles per plant. The genotype Kavunginpoothala showed maximum days for 50% flowering (132) followed by Chenkayama (121), which was on par with Kavuni (120). The study on physiological aspects showed that the highest net photosynthetic rate at reproductive stage was exhibited by Kala Namak (28.73 µmol CO2 m-2 s -1 ) followed by Kothambalarikayama (28.33 µmol CO2 m-2 s -1 ) and the lowest was shown by Japan Violet (20.87 µmol CO2 m-2 s -1 ). Chempav possessed highest chlorophyll content (3.25 mg g-1 ) which was on par with Chenkayama (3.20 mg g-1 ) and the lowest chlorophyll content was observed in Japan Violet (1.00 mg g-1 ). Highest IAA content at reproductive stage was observed for Kavunginpoothala (873.60 µg of unoxidized auxin g-1 h-1 ) and Ithikandapan (354.87 µg of unoxidized auxin g-1 h1 ) exhibited the lowest IAA content. Irrespective of growth stages, Kavunginpoothala recorded maximum GA content (18.99 mg/g, 20.82 mg/g, 20.87 mg/g). In maturity stage, Ithikandapan (12.69 mg/g) had lowest GA content. Thavalakkannan and Chenkayama, showed a purple coloured stem during reproductive stage. At maturity, Black Jasmine, Ramli, Kala Namak, and Karutha Modan exhibited a black coloured husk while remaining all other genotypes were in brown colour. Among the entries, grains for nearly nineteen are red pigmented; five are white pigmented and one being black pigmented. The highest average panicle weight was possessed by Chuvanna Vattan (18.51 g) followed by Rakthasali (18.35 g). The lowest average panicle weight was for Chenkayama (10.36 g), which was on par with Kavunginpoothala. The local check Uma and Jyothi exhibited an average panicle weight of 27.46 g and 25.64 g, respectively. Chuvanna Vattan (26.68 g) exhibited the maximum 1000 grain weight compared to other genotypes, followed by Njavara (26.26 g). The lowest 1000 seed weight was observed in Rakthasali (16.69 g). The local check Uma and Jyothi had 1000 weight of 27.46 g and 28.49 g, respectively. Black Jasmine possessed the significantly high iron (39.65, 24.71 mg/kg) and zinc (19.21, 17.67 mg/kg) contents in unpolished and polished grains. The anthocyanin content was found high in Black Jasmine (1110.05 µg/g), followed by red variety Rakthasali (1058.49 µg/g) and Njavara (1001.51 µg/g). The nutraceutical components such as gamma oryzanol, tocopherol, total polyphenols and flavonoids was significantly high in Black Jasmine (72.85 mg/100 g, 5.32 µg/g, 13.13 mg/g and 6.46 mg/g, respectively) followed by Rakthasali (63.12 mg/100 g, 4.33 µg/g, 8.89 mg/g, and 6.25 mg/g, respectively) and Njavara (61.80 mg/100 g, 4.26 µg/g, 8.77 mg/g and 6.17 mg/g, respectively). The cooking and eating quality of rice was determined by the proximate content and carbohydrate digestibility. Among the studied genotypes, significantly highest ash (2.53%), fibre (2.43%), fat (2.64%), lowest digestibility (60%) and high antioxidant activity were observed in Black Jasmine. It was also observed that, polishing caused a reduction in bioactive contents. Due to polishing, nearly 80 and 50 per cent reduction was found in mineral content and antioxidant activity. Among the selected genotypes, it could be noted that, black pigmented landraces exhibited significantly high bioactive compounds followed by red and white. Thus, based on the consistency in bioactive compounds, HPLC characterization was also carried out at CSIR-CFTRI, Mysore for a subset of the major compounds to identify and quantify them in nine selected representatives from the red, black and white rice types. The HPLC analyses indicated that Rakthasali had the highest chlorogenic acid (14.27 mg/100 g) content, while Black jasmine had high gallic acid (3.56 mg/100 g). Njavara showed highest t-cinnamic acid (1.47 mg/100 g) content compared to other varieties. These sub compounds may be the reason for high nutraceutical potential these pigmented genotypes. It was also noted that white genotypes had the presence of many sub compounds, even though white rice is considered inferior to black and red. In the next phase of the study, the three best promising genotypes (Rakthasali, Black Jasmine and Njavara) were grown in field during Kharif and Rabi. The field experiments were laid in randomised block design at RARS, Pattambi, with an aim to analyse the phenolic content, anthocyanin content and antioxidant activity. The results indicated that, the grains harvested from the Kharif season exhibited high phenolic and anthocyanin contents along with high antioxidant activity compared to the Rabi season crop. Subsequently, all the grains were subjected to storage (ageing) study to check the bioactive compound activity during storage. The grains were stored under ambient conditions and bioactive profiling (total phenolics, anthocyanin, antioxidant activity) was carried out in grains at harvest, 6, 9 and 12 months after storage. A tremendous reduction was observed in all the grains stored. Among the genotypes, Rakthasali, Thavalakkannan and Kattamodan exhibited lowest reduction in phenolic content (~30 %). Black Jasmine, Japan Violet and Chenkayama had lowest reduction in anthocyanin content (~ 15 %). Rakthasali, Karutha Modan, Krishnakaumudh and Njavara showed lowest reduction in antioxidant activity (~ 30 %) among the selected entries. Simultaneously, an in silico study was conducted on CB dock platform to find out the best bioactive compound against receptors of major lifestyle diseases such as cancer, diabetes and inflammation, based on the interaction dynamics. The major sub-compounds of phenolics, flavonoids, oryzanols, anthocyanins and tocols were selected for the docking study. The disease specific target proteins/receptors were selected for performing the docking assay. Chlorogenic acid was identified as best phenolic sub-compound against cancer, diabetes and inflammation. Among the oryzanol sub-compounds, stigmasteryl ferulate, sitostanyl ferulate and cycloartenyl ferulate exhibited best interaction with receptors, evidenced by superior docking scores. Among flavonoids, luteolin, myrcetin and fisetin are the promising drug candidates against these diseases. In case of tocols, strong interaction with target proteins was noted for gamma toco-trienol, beta toco-trienol and delta toco-trienol. delphinidin-3-O-galactoside, and cyanidin-3-O-glucoside are the major sub compounds of anthocyanin which showed best docking scores with selected target proteins. From the present study, based on the docking scores and ADMET properties, the above selected sub compounds which were also pronounced in Rakthasali, Black Jasmine and Njavara as per their HPLC profiles, were found promising to combat lifestyle disorders Further validation by wet lab studies is required to reinforce their promising roles. The study describes rice bran is the main source of minerals, vitamin E and other bioactive compounds known to possess medicinal properties. The study highlights the nutraceutical potential of Rakthasali, Black Jasmine and Njavara and the possibilities for exploiting them by selecting the apt growing season, ageing stage and targeted extraction of the relevant bioactive compound (s), for reaping maximum benefits. The relevance of further examination of these varieties for their stress resilience and marginal yield increase potential also is a future thrust.