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 Management of banana bract mosaic virus using beneficial fungal root endophyte, Piriformospora indica(Department of Plant Pathology, College of Agriculture ,Vellayani, 2024-03-20) Sinijadas, K.; Joy, MThe research work entitled “Management of Banana bract mosaic virus using beneficial fungal root endophyte, Piriformospora indica” was carried out at the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during the academic year 2018-2022. The objective was to evaluate the beneficial fungal root endophyte, P. indica for the management of Banana bract mosaic virus (BBrMV); and to elucidate the role of antioxidants and plastids in this tripartite interaction. The characteristic symptoms of BBrMV viz., reddish spindle shaped streaks on pseudostem, black necrotic streaks on peduncle, chlorotic spindle lesions on leaves and underdeveloped fruits were observed during the survey conducted in five agro-climatic zones (ACZ) of Kerala. Percent disease incidence (PDI) and vulnerability index (VI) of the disease recorded from different zones showed the highest in banana var. Nendran (PDI - 61.66 & VI – 44.03) from southern zone and lowest in var. Poovan (PDI – 7.29 & VI – 6.29) in central zone. Serological and molecular detection confirmed the presence of BBrMV in banana var. Nendran. The sequence similarity analysis of the coat protein (CP) gene of BBrMV southern zone isolate (Vellayani) showed 99.12 percent genetic closeness to its Tamil Nadu isolates compared to the Kerala isolates (98.38 percent). Screening of the most virulent strain of BBrMV from five different ACZ of Kerala was done based on early symptom development in TC banana plants var. Nendran. The BBrMV strain from southern zone could produce the symptoms on var. Nendran within 30 days (lowest) followed by central zone (55 days) on artificial inoculation with viruliferous aphids, Pentalonia nigronervosa. The cross-infection study of virulent strain of BBrMV (southern zone) of Nendran on Nendran developed chlorotic spindle lesions on leaves at 30 days after inoculation followed by Nendran on Robusta (75 days). P. indica-colonization in banana var. Nendran was carried out using standardized medium. The chlamydospores of the fungus were observed in roots at 20 days after colonization (DAC). In both in vitro and in vivo experiments, P. indicacolonized plants showed reduced disease severity irrespective of the virusinoculation stage with a vulnerability index of 6.7 percent in BBrMV (+Pi / +V); and 20.0 percent in the virus-infected plants post-colonized with P. indica (+V / +Pi) compared to 53.3 percent in the virus alone infected plants. Further, PCR analysis with BBrMV coat protein specific primer yielded amplicon of low intensity in P. indica-colonized plants inoculated with the virus compared to the control plants indicating the ability of the fungus to inhibit the virus. Further, P. indica precolonized plants inoculated with BBrMV had improved growth and yield parameters compared to non-colonized plants. Field trial was laid out with two treatments (P. indica-colonized and noncolonized banana plants var. Nendran) at Instructional Farm, College of Agriculture, Vellayani. P. indica-colonized plants recorded a drastic reduction in the severity of BBrMV by 33 to 58 percent compared to non-colonised control plants. Enhanced plant height (30 percent), collar girth (45 percent), number of leaves (25 percent), leaf length (30 percent), leaf width (27 percent), fresh weight of shoot (34 percent), number of secondary roots (62 percent), number of tertiary roots (76 percent) and root weight (86 percent) were observed in P. indica-colonized plants at 90 days after treatment. P. indica-colonization also improved the bunch weight (32.9 percent) and fruit quality. Biochemical detection of superoxides using nitroblue tetrazolium (NBT) and H2O2 with diamino benzidine (DAB) stains at 5, 10, 15, 30 and 45 days revealed a reduction in reactive oxygen species (ROS) accumulation in both P. indica-colonized plants challenged with BBrMV (+Pi / +V) and virus infected plants post-colonized with P. indica (+V / +Pi) compared to BBrMV alone. The decrease in ROS production and disease severity in the endophyte-colonized plants inoculated with the virus were attributed to the increased activities of antioxidant enzymes viz., peroxidase, superoxide dismutase, catalase, glutamate synthase and ascorbic acid oxidase. The molecular analysis of genes involved in the symptom development indicated the beneficial effect of P. indica on BBrMV infection in banana. P. indica reduced the symptoms by up-regulating chlorophyll biosynthesis gene (chlorophyll synthase-CHLG) and down-regulating chlorophyll degradation genes (chlorphyllase CLH1 & CLH2; and pheophytin pheophorbide hydrolase - PPH), carotenoidbiosynthesis genes (phytoene synthase-PSY1 and PSY2), carotenoid degradation gene (Phytoene desaturase - PDS) and the virus specific genes responsible for symptom development (Hc-Pro and P3). Thus, the present study reveals that P. indica enhances tolerance against BBrMV in addition to improved growth promotion, yield and fruit quality in banana plant.Item Piriformospora indica and its water diffusible exudates for the management of chilli anthracnose incited by colletotrichum capsici (Syd.) butler and bisby(Department of Plant Pathology, College of Agriculture, Vellayani, 2024-07-05) Elizabeth, T Jojy; KAUThe research work entitled “Piriformospora indica and its water diffusible exudates for the management of chilli anthracnose incited by Colletotrichum capsici (Syd.) Butler and Bisby” was carried out in the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during 2017- 2023. The study was undertaken with the objective to evaluate P. indica- and its water diffusible exudates- primed chilli seedlings and plants against foliar infection of C. capsici; and study the biochemical and molecular mechanisms involved in this tripartite interaction. A survey conducted in the five agroclimatic zones (ACZs) of Kerala showed that the highest disease incidence (DI) and Percent Disease Index (PDI) were recorded at RARS, Pilicode (DI-90 & PDI-52.60) of northern zone, while the lowest DI was observed at farmer’s field, Kottarakkara (20%) and ORARS, Kayamkulam recorded the lowest PDI (23.63). Chilli anthracnose symptoms namely leaf spot, leaf blight, die-back, fruit rot and mummified fruits were observed in different survey locations of the five agroclimatic zones (ACZ) of Kerala. Nine C. capsici isolates and one isolate of C. gloeosporioides were obtained from the surveyed locations. All the C. capsici isolates produced sparse mycelial growth with concentric zonations of acervuli on potato dextrose agar (PDA) medium. The upper side of culture plates appeared in different shades of white and off-white to grey with regular or irregular margins, while the reverse side looked yellowish brown to black. Isolate Cc4 produced maximum mycelial growth diameter of 8.6 cm compared to 7.2 cm (minimum) in Cc2 on 7th day after inoculation (DAI). Further, microscopic characters such as mycelial width, size of conidia, acervuli, appressoria, number and length of setae were significantly different in the C. capsici isolates. The most virulent isolate of C. capsici was screened based on the lesion size produced on the artificially inoculated leaves and fruits of chilli var. Vellayani Athulya. On 7th DAI, Cc3 isolate of C. capsici produced maximum lesion size of 2.52 cm while isolate Cc4 produced minimum lesions (0.74 cm) on 292 inoculated chilli leaves. Similarly, tender, mature and ripe fruits of var. Vellayani Athulya inoculated with Cc3 isolate produced the highest lesion size (2.48, 2.34 and 2.56 cm respectively) and isolate Cc4 recorded the lowest lesions (1.62, 1.16 and 1.38 cm respectively) on 7th DAI. Thus, isolate Cc3 from Thrissur, was selected as the most virulent isolate of C. capsici. Ten selected KAU/ICAR released varieties of chilli were screened with Cc3 isolate by detached leaf and fruit inoculation method to identify the most susceptible variety. Leaves of Vellayani Athulya recorded the highest lesion size of 2.80 cm on 5 DAI compared to 1.66 cm (lowest) in Manjari. Maximum lesion size of 2.96 cm was observed on the tender fruits of Vellayani Athulya and minimum was recorded on Vellayani Thejus (1.52 cm). Lesions of 3.50 cm (highest) were observed on mature Vellayani Athulya fruits as against 2.00 cm on Manjari fruits. Similarly, ripe fruits of Vellayani Athulya recorded maximum lesion size of 3.46 cm and minimum in Vellayani Samrudhi (1.30 cm) at 7 DAI. Also, in the in vivo study, higher lesion size (3.00 cm) was observed in Vellayani Athulya fruits and lowest (1.62 cm) in fruits of Vellayani Samrudhi at 7th DAI. Standardization of P. indica-colonization in chilli var. Vellayani Athulya was done on plant nutrient medium (PNM) and chilli roots were sampled at different intervals viz., 3rd, 5th, 7th, 10th, and 15th days after colonization (DAC). Young, double-walled chlamydospores were observed within the root cells at 5 DAC. P. indica colonization gradually increased and reached a maximum of 100 per cent at 15 DAC. Dual culture of P. indica and C. capsici showed the appearance of inhibition zone (8.33 DAI) and antibiosis (12.67 DAI) at the point of interaction of the two fungi. Further microscopic observations revealed thickening and lysis of pathogen mycelium by P. indica. The growth of C. capsici was suppressed (3.85 cm) in dual culture compared to normal (6.28 cm) in control at 7th DAI. 57.22 per cent mycelial growth inhibition due to P. indica was observed at 15 DAI. Antagonistic property of P. indica-water diffusible exudates (Pi-WDE) against C. capsici was studied by in vitro poison food technique. Significant 293 differences were observed in the colour and nature of C. capsici growth on PDA media, added with different day-old Pi-WDE in comparison with control. Among them, ten day-old Pi-WDE recorded the highest inhibition (67.30 %) of pathogen mycelial growth whereas the lowest in one day-old Pi-WDE (9.29 %). Mass multiplication of P. indica in the portray medium was carried out. Rapid growth of the endophyte was recorded in a combination of farmyard manure (FYM) and coir pith (1:1), added with 2 per cent gram flour. P. indica completely covered the inoculated trays within 7 DAI. This medium was used to colonize chilli plants with P. indica for further experiments and thus, four treatments viz., P. indica alone (T1), C. capsici alone (T2), P. indica-primed seedlings + C. capsici (T3) and control (T4) were systematized for the in vitro and in vivo evaluation experiments. In vitro study of P. indica-primed chilli seedlings against C. capsici significantly reduced anthracnose lesion size and disease severity to 1.30 cm and 30.63 per cent respectively compared to 2.06 cm and 77.00 per cent in C. capsici alone at 7 DAI. Maximum root and shoot weight were recorded in the P. indicacolonized plants, irrespective of the pathogen. Pot culture experiment was also carried out in the chilli var. Vellayani Athulya. The development of anthracnose symptoms on leaves was delayed by 4 days in P. indica-colonized chilli plants as against 2 days in control. The lesion size and disease severity due to anthracnose was lower in P. indica-colonized chilli leaves (1.58 cm & 54.90 %) compared to C. capsici alone (2.86 cm & 85.20 %) at 10 DAI. Similarly, minimum lesion size and disease severity of 1.17 cm and 27.92 per cent was observed in fruits from pathogen inoculated P. indicacolonized chilli plants than in C. capsici alone (3.56 cm & 91.67 %) at 10 DAI. Field studies were conducted in rabi and summer seasons with two treatments viz., P. indica-colonized and non-colonized plants. P. indica-colonized chilli plants recorded lower anthracnose severity of 27.00 per cent in Rabi and 15.50 per cent in summer compared to 56.50 and 47.00 per cent respectively in control. P. indica colonization improved the biometric characters such as plant height, number of leaves, leaf area, number of secondary and tertiary roots, shoot 294 weight, root weight in primed chilli plants. In addition, yield of chilli was enhanced in endophyte-colonized plants compared to control. Biochemical studies on the mechanism of disease management in P. indica-colonized chilli plants at 0, 12, 24 and 72 hours after inoculation (HAI) showed increased activity of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (PO). In contrast, the activities of 4-coumaryl CoA ligase (4-CL) and polyphenol oxidase (PPO) were enhanced in chilli plants inoculated with C. capsici alone. Isoenzyme analysis revealed higher induction of isoenzymes of PO and PPO in the P. indica-colonized plants compared to control. Enhanced induction of pathogenesis related (PR) proteins was noticed in the P. indica-colonized plants inoculated with the pathogen at 72 HAI. The capsaicin content was drastically reduced in C. capsici inoculated chilli fruits. However, fruits from P. indica-colonized plants had high capsaicin content (>30% over control) irrespective of infection. Molecular studies of the defense related genes revealed the positive role of P. indica in the management of anthracnose disease in chilli. P. indica reduced anthracnose symptoms in colonized chilli plants by the upregulation of genes involved in phenyl propanoid pathway (PAL1, 4-CL, CAD and CHS), jasmonic acid signaling pathway (PDF1.2 and AOS) and salicylic acid signaling pathway (PR1, EDS1 and PAL3); and downregulating the LOX gene involved in jasmonic acid signaling. In summary, P. indica and its WDE inhibited C. capsici under in vitro conditions. Anthracnose incidence and severity were considerably reduced in the treated chilli seedlings and plants compared to control. In addition, P. indica enhanced growth as well as yield in colonized chilli plants, thereby enhancing the fruit quality. Further, the endophyte improved disease resistance in chilli plants by increasing the defense-related enzyme activities and expression of defense genes. Thus, the root fungal endophyte, P. indica can be considered as an efficient biocontrol agent in the management of chilli anthracnose.