1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
Permanent URI for this communityhttp://localhost:4000/handle/123456789/1
Browse
14 results
Search Results
Item Piriformospora indica- mediated degradation of strobilurin and triazole fungicides in tomato plants(Department of Plant Pathology, College of Agriculture,Vellayani, 2025) Austin Raj, K S.; Joy, MThe research work entitled “Piriformospora indica-mediated degradation of strobilurin and triazole fungicides in tomato plants” was carried out at the Department of Plant Pathology, College of Agriculture Vellayani, Thiruvananthapuram during 2022-24. The objective was to evaluate the beneficial fungal root endophyte, P. indica on degradation or retention of strobilurin and triazole fungicides used for the management of black leaf mold of tomato. P. indica-colonization in tomato var. Vellayani Vijai was done in the standardized medium. The presence of the chlamydospores was observed in roots of tomato at 5 days after colonization (DAC). P. indica-colonization was further confirmed using fungus-specific primer Pi-tef by PCR. Compatibility of P. indica was studied with fungicides such as azoxystrobin, pyraclostrobin, propiconazole and tebuconazole. P. indica is compatible with azoxystrobin and to some extend with pyraclostrobin; and incompatible with propiconazole and tebuconazole. P. indica exhibited 72 per cent compatibility with azoxystrobin at 50 ppm; while 19.72 per cent compatibility was observed with pyraclostrobin. P. indica could not grow in triazole fungicides, propiconazole and tebuconazole even at 50 ppm. Leaves with characteristic symptoms of black leaf mold of tomato were collected from Coconut Research Station, Balaramapuram. The symptoms were yellow and chlorotic spots on the upper leaf surface and black sooty mold on the corresponding lower surface. The fungus was isolated on potato dextrose agar (PDA) medium. The mycelial growth was grey on the upper side with concentric zonations, and black on the rear side. The margin of the growth was regular. The growth rate was observed as 1.1 mm per day. Koch’s postulate of the pathogen was proved by detached leaf assay. Symptoms started appearing from 5th day onwards. Symptoms were chlorotic lesion on the upper surface and black sooty growth on the lower surface of the leaf. In vitro evaluation of the pathogen against azoxystrobin, pyraclostrobin, propiconazole and tebuconazole was done by poisoned food technique. Both strobilurin and triazole fungicide significantly inhibited mycelial growth of 165 Pseudocercospora fuligena. Triazole fungicides such as propiconazole and tebuconazole were more effective in inhibiting the mycelial growth of P. fuligena than strobilurin fungicides. At 50 ppm, P. fuligena has grown 3.80 cm in azoxystrobin, 3.80 cm in pyraclostrobin, 1.37 cm in propiconazole and 1.21 cm in tebuconazole fungicides. P. fuligena growth was completely inhibited at 100 ppm of triazole fungicides; but at least 25 per cent fungal growth was observed in strobilurin fungicides even at 1000 ppm. A field experiment was conducted during the summer season (2023) at Coconut Research Station Balaramapuram. The first spray of fungicides was given at the vegetative stage ie., 40 days after sowing. The second spray of fungicides was given after fruit set stage ie., 70 days after sowing. The leaves and fruit samples were collected at 2 h, 24 h, 3, 5 and 10 days after spraying and residues were assessed by LC-MS/MS. P. indica enhanced the retention of the fungicide residues in leaves. The fungus also enhanced the degradation of the fungicides in tomato fruits which makes the fruits safer to eat. P. indica-colonization decreased the incidence and severity of diseases such as black leaf mold, Alternaria leaf spot, leaf curl and mosaic under natural incidence. When azoxystrobin was used, 64.05 per cent reduction of black leaf mold severity was observed over control at 60 days after transplanting. When propiconazole was used, 55.49 per cent reduction was observed. When azoxystrobin was used along with P. indica, the disease severity has been further reduced to 71.89 per cent; and when propiconazole was used along with P. indica, a reduction of 69.25 per cent was observed. Moreover, P. indica enhanced the growth parameters such as plant height, number of flowers per plant, number of fruits per plant and average yield per plant. Further P. indica decreased the number of days taken for flowering, 50 per cent flowering, fruit set and 50 per cent fruit set. Molecular basis of P.indica-mediated degradation of strobilurin and triazole fungicides was done at 3 and 7 days after the application of the fungicides. RNA was extracted and reverse transcribed into cDNA. GAPDH was used as the house keeping gene. Expression of genes involved in the degradation of strobilurin fungicides such as strH and Carboxyl esterase and Ergosterol gene involved in the degradation of triazole were studied. P. indica-colonization significantly enhanced the expression of Carboxyl esterase and Ergosterol genes in the fruits, thus increasing the fungicides degradation in fruits; whereas it repressed the expression of strH and Ergosterol genes 166 in leaves, thereby increasing the retention of fungicides in leaves under field conditions. Relative expression of the genes at 7th day after the application the fungicides was less compared to that at 3rd day. P. indica-colonization together with triazole or strobilurin fungicides application could effectively control the black mold disease caused by P. fuligena with enhanced biometric and yield characters of tomato. P. indica-colonization significantly enhanced the retention of the fungicides in leaves by down regulating the fungicide degrading genes; and degradation of fungicides in fruits by upregulating the fungicide degrading genes under field conditions with ecological sustainability and also to produce safe-to-eat tomatoes.Item Biotization using piriformospora indica for induction of in vitro floral primordia in saffron(crocus sativus L.)(Department of molecular biology and biotechnology, College of agriculture , Vellayani, 2023-09-25) Midhukrishna; Smitha BhasiThe study entitled "Biotization using Piriformospora indica for induction of in vitro floral primordia in saffron (Crocus sativus L.)" was conducted at the Department of Molecular Biology and Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram during 2022-23. The objective of the study was to evaluate the effect of biotization using Piriformospora indica for culture establishment and induction of in vitro floral primordia in saffron. The explants namely corms collected from farmer’s field, Jammu were used for the study. The pure cultures of P. indica were collected from the Department of Agricultural Microbiology, College of Agriculture, Vellayani. The effect of biotization of corms using P. indica was evaluated both in potting medium (Treatment 1) as well as in Murashige and Skoog (MS) medium (Treatment 2). Non-biotized corms were used as control (Treatment 3). Corms were planted in sterile potting medium (perlite and compost in 1:1 ratio) during June and kept in shade conditions for 3 months. However, no sprouting or root induction was noticed and later the corms got dried due to fungal contamination. Another strategy attempted was inducing in vitro rooting and further biotization. For this, the corms were kept at 16ºC during August-October. 100% sprouting was observed in 50 days of treatment and the sprouts thus induced in vivo were maintained at 18ºC for elongation. Further, 120-day-old elongated sprouts were cultured in basal MS medium for root induction. Healthy roots appeared in 3 days and in vitro rooted plants were transferred to potting media incorporated with P. indica mycelium. However, roots of all samples got dried in a week. For attempting biotization in vitro, full corms and 1cm3 cut corms were used for initiating sterile cultures. Sprouts were initiated within 15 days from 1cm3 cut corms cultured in MS medium supplemented with 1.5ppm BAP (6 benzylaminopurine). Further, the sprouts induced both in vitro and in vivo were subjected for elongation in basal MS medium. Significant increase in shoot length was noticed in sprouts induced in vivo (kept at 16ºC) compared to sprouts induced in vitro. Flower buds were induced from elongated sprouts by sub culturing in MS 81 medium and 50% sprouts that were induced in vivo, developed in vitro floral buds. However, no floral buds were observed in elongated sprouts induced in vitro. Biotization of in vitro cultures (Treatment 2) was carried out using P. indica agar discs and derivatives of P. indica. 130-day-old elongated sprouts having in vitro roots were inoculated with P. indica agar discs in MS medium for colonization. However, colonization was not observed under microscopic analysis. 165-day-old elongated sprouts were also treated with derivatives of P. indica and compared with control. Elongated sprouts treated with derivatives of P. indica exhibited a significant increase in plant height compared to control. These sprouts were further sub-cultured for a period of 7 weeks in MS medium for inducing multiple shoots. 165-day-old elongated plantlets treated for 80 days with derivatives of P. indica produced more number of multiple shoots per corm compared to control. Further, the multiple shoots developed were cultured in MS medium supplemented with 9% sucrose for inducing in vitro cormlets. Multiple shoots pre-treated with P. indica produced larger and more number of cormlets compared to control. To conclude, sterile potting media (perlite-compost) was found not suitable for the establishment and growth of corms under field conditions of Vellayani region. Storage of corms at 16ºC during dormant stage is found critical for in vitro growth, rooting and flowering in saffron. In vitro treatment using derivatives of P. indica in saffron showed positive responses with respect to plant height, number of multiple shoots, number, and size of in vitro cormlets. Hence, derivatives of P. indica can be used for better in vitro establishment of cultures, multiple shooting, and in vitro cormlet production in saffron.Item Role of phytohormones and oxidative stress in piriformospora indica mediated tolerance to ralstonia solanacearum inciting bacterial wilt of tomato(Department of Plant Pathology, College of Agriculture, Vellayani, 2023-11-07) Ruby Nahan, P.; Sajeena, AThe study entitled “Role of phytohormones and oxidative stress in Piriformospora indica mediated tolerance to Ralstonia solanacearum inciting bacterial wilt of tomato” was conducted during 2019-2022 at College of Agriculture, Vellayani and Integrated Farming System Research Station (IFSRS), Karamana with an objective to evaluate the beneficial fungal root endophyte, Piriformospora indica for the management of Ralstonia solanacearum inciting bacterial wilt of tomato and elucidating the role of phytohormones and oxidative stress in the tripartite interaction. Ralstonia solanacearum, the causative agent of bacterial tomato wilt, was isolated from the stem of an infected tomato plant showing typical wilt symptoms collected from a farmer’s field at Nanniyod, Thiruvananthapuram and tested for the presence of ooze for confirmation. The disease symptoms were initial drooping of leaves followed by sudden wilting of whole plants and brown discoloration of vascular region. A white fluidal colony with a red center was obtained on the triphenyl tetrazolium chloride (TZC/TTC) medium and pathogenicity was tested by two methods viz., soil drenching and root dip method to prove Koch's postulates. The symptoms were observed within 5 days after artificial inoculation and complete wilting was observed in 8, 10 days in root dip and soil drenching respectively. Morphological studies revealed R. solanacearum as gram negative and rod-shaped. By choosing the most virulent colony and diffusing it in distilled sterile water, the bacteria might be preserved for a long time. The maintained culture exhibited virulence up to 5 months. P. indica , the beneficial root endophyte was grown on potato dextrose agar (PDA) medium, On 9 days following inoculation, the fungus had grown completely in a petri-plate (9 cm dia.). The fungus grows into a mat in potato dextrose broth (PDB) after 16 days of inoculation. The fungal mat was transferred to a sterilized potting mixture and under sterile circumstances; the fungus reached full growth in one week. Tomato seedlings were co-cultivated with P. indica in a pro tray mixture made up of coir pith and dried farm yard manure (FYM) with two per cent gram flour. The roots were examined under a microscope at 10, 15, 20, 25 and 30 days after co-cultivation (DAC) and the presence of fungal spores was seen. The spore size grew over time. P. indica colonization reduced the time for seeds to germinate by two days compared to the control and increased the germination percentage 21 per cent over the non-colonized seeds. An in vivo study was undertaken at IFSRS, Karamana for the management of bacterial wilt disease in tomato (var. Pusa Ruby) by P. indica-colonised seedlings inoculated with R. solanacearum at 0 days after colonization (DAC), 2 DAC, 5 DAC, 10 DAC and P. indica-colonised seedlings, Seedlings inoculated with R. solanacearum, R. solanacearum inoculated seedlings drenched with copper hydroxide @ 2g L-1, uninoculated non colonized seedlings as checks. Seedlings inoculated with R. solanacearum and P. indica-colonised seedlings inoculated with R. solanacearum at 0 days after colonization revealed 100 per cent disease incidence whereas, P. indica-colonised seedlings inoculated with R. solanacearum at 2 DAC, 5 DAC, 10 DAC showed no disease incidence (0 %). The treatment P. indica-colonised seedlings inoculated with R. solanacearum 10 DAC revealed the minimum number of days taken for flowering (35 DAT), fruit weight (41.80 g), yield per plant (590 g), leaf area (2.0 cm2), number of leaves (26), root length (27 cm) and plant height (25.2 cm) followed P. indica-colonised seedlings inoculated with R. solanacearum 5 DAC with number of days taken for flowering (36 DAT), fruit weight (43.93 g), yield per plant (620 g). The treatments P. indica-colonised seedlings inoculated with R. solanacearum at 2 DAC, 5 DAC and 10 DAC were effective than P. indica non-colonized seedlings. The colonization of P. indica has significant effect on the growth and development of the tomato plant. Field study was conducted at College of Agriculture, Vellayani and Integrated Farming System Research Station (IFSRS), Karamana. Bacterial wilt incidence in two treatments viz., P. indica-colonized and P. indica non-colonized was studied. Both the treatments were got infected by R. solanacearum under natural condition but P. indica-colonized seedlings could prolong the longevity of tomato seedlings (var. Pusa Ruby) for 5 days compared to P. indica non-colonized seedlings. P. indica colonization has significantly reduced the number of days taken for flowering (20 DAT) and increased the plant growth parameters viz., plant height (31.74 cm), number of leaves (25), root length (26.44 cm), leaf area (4.89 cm2). According to the study, P. indica colonization plays a key role in the growth and development of tomato plants, but its colonization alone is ineffective against bacterial wilt disease of tomato (var. Pusa Ruby) in a sick plot condition. Biochemical studies on identifying the mechanism of disease tolerance P. indica colonized plants were studied at 18 DAS and 20 DAS. P. indica colonized plants challenged with R. solanacearum showed highest activity of the enzymes viz., PO, PPO, PAL, SOD, catalase, IAA and GA. P. indica colonized plants showed reduction in the ROS production. Thus, the present study revealed that bacterial wilt of tomato caused by R. solanacearum is an important disease. The colonization of P. indica in tomato (Var. Pusa Ruby) roots significantly improved a number of plant development characteristics, but its colonization alone was unable to control bacterial wilt. Therefore, from the study it is inferred that the management of bacterial wilt disease of a highly susceptible variety of tomato Pusa Ruby in a heavily infested soil is not possible by P. indica colonization.Item Standardization of propagation and performance assessment of mechanical harvesters in clove (Syzigium aromaticum (L) Merr. & Perry)(Department of Plantation Crops and Spices, College of Agriculture, Vellayani, 2023-05-08) Rejith, S; Sreekala, G SThe present investigation entitled “Standardization of propagation and performance assessment of mechanical harvesters in clove (Syzigium aromaticum (L) Merr. & Perry).” was carried out in the Department of Plantation Crops and Spices, College of Agriculture, Vellayani during the period 2020-2022 with the objective of standardization of seed treatment for improved seed germination, grafting method and performance assessment of mechanical harvesters in clove. Clove is commercially propagated by seeds but the viability of the clove seed is less and germination is generally poor. Hence, to improve the seed germination rate an experiment was conducted using seven seed pretreatments and were compared with the control. Freshly harvested fully matured clove fruits are collected and subjected to different pretreatments viz. IAA 20 ppm (T1), GA3 50 ppm (T2), GA3 100ppm (T3), PGPR Mix Ⅰ 5 per cent formulation (T4), PGPR Mix II 5 per cent formulation (T5), Piriformospora indica 105cfu/ml (T6), carbendazim 0.1% (T7) and control (T8). These pretreated seeds were sown in protrays and kept under two growth condition viz. polyhouse with temperature and humidity control (C1) and polyhouse without temperature and humidity control (C2). T1 C1 recorded the highest germination and survival percentage of 90% at 3MAS, basal shoot girth of 0.42cm, mean germination time of 33.15 days, mean germination rate of 3.02 x10-2day-1, coefficient of velocity of germination of 3.21x10-2, germination rate index of 26.82 %/day, germination index of 1477.30, root shoot ratio of 0.50 and allometric index of 0.99. At the same time T2C1 recorded the highest number of leaves of 7, leaf length of 6.90cm, leaf breadth of 2.76cm, leaf area of 12.37cm2, shoot length of 15.51cm, fresh shoot weight of 2.28g, dry shoot weight of 0.48g, root length of 13.85cm, fresh root weight of 0.43g, dry shoot weight of 0.17g, seedling vigour index Ⅰ of 2598.26 and seedling vigour index Ⅱ of 55.02. Even though seed treatment of IAA 20ppm along with growth condition in Polyhouse with temperature and humidity control was identified as ideal for better seed germination, seed treatment of GA3 50ppm along with growth condition in polyhouse with temperature and humidity control showed superior seedling growth characters. 138 Clove is commercially propagated by seed and development of any vegetative method of propagation can be helpful in reducing the height of the crop, early yield and true to type. The percentage of success to propagate clove by vegetative method was less. The production of phenol content may be one reason for this. Hence methods to overcome the phenol content was tried along with different grafting methods. The treatments included approach grafting without antioxidant treatment (T1), approach grafting with antioxidant treatment (T2), soft wood grating without antioxidant treatment (T3), soft wood grafting with antioxidant treatment (T4), epicotyl grafting without antioxidant treatment (T5), epicotyl grafting with antioxidant treatment (T6), patch budding without antioxidant treatment (T7), patch budding with antioxidant treatment (T8) kept under two growth conditions viz. polyhouse with temperature and humidity control (C1), polyhouse without temperature and humidity control (C2). At the end of 6 months, T2C1 recorded higher number of leaves (13.30), length of leaves (4.51) and breadth of leaves (2.46). Hence approach grafting with antioxidant treatment with growth condition of Polyhouse with temperature and humidity control is found to be best for the vegetative propagation of clove Performance assessment of mechanical harvesters and manual harvesting in clove were analysed. Mechanical harvesters such as VIMAL 2 stage tree pole pruner (T1), Wolf-Garten Bypass steel Telescopic pruner (T2), SHAFIRE Steel Blade Hand Pruner (T3) and Manual harvesting(T4) were compared. Manual harvesting was found to be more economically feasible, taking only less time for postharvest cleaning operation with significantly lower operator fatigue and high field coverage.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.Item Evaluation of beneficial fungal root endophyte, Piriformospora indica for the management of Tomato leaf curl virus(Department Of Plant Pathology College Of Agriculture, Vellayani, 2021-12-30) Saru Sara Sam; Joy, MThe research work entitled “Evaluation of beneficial fungal root endophyte, Piriformospora indica for the management of Tomato leaf curl virus” was done at the Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during the academic year 2019-2021 with the objectives of evaluation of the beneficial fungal root endophyte Piriformospora indica for the management of Tomato leaf curl virus (ToLCV) in tomato; and elucidating the role of reactive oxygen species (ROS) and antioxidant enzymes in the tripartite interaction. ToLCV symptoms were assessed and maintained in insect proof nets from the various field at College of Agriculture, Vellayani, Thiruvananthapuram. The major symptoms observed includes mild curling with yellow speckles, severe leaf curling with blisters on leaf, stunted growth and reduced leaf size with flower abortion. Tomato variety Vellayani Vijay showed a disease incidence of 72.3 to 100 per cent with vulnerability index of 68.5 to 71.0 at different fields, while Anagha variety recorded an incidence of 67.0 per cent and vulnerability index of 54. The presence of ToLCV was confirmed using the universal primers viz., Deng and AV/AC that amplifies the coat protein of Begomovirus which produce amplicons of 520 bp and 575 bp respectively under specified conditions. P. indica-colonization in tomato variety Vellayani Vijay was done in portray mixture amended with 2 per cent gram flour. P. indica spores were observed from 3 day after colonization (DAC) and the spore size increased by subsequent days. P.indica-colonization enhanced the growth parameters including plant height, shoot height, and root and shoot biomass. The tomato plants colonized with P. indica showed an increase in shoot height, number of branches, number of leaves, number of flower, fresh shoot weight, dried shoot weight, fresh root weight and dried shoot weight by 34, 117, 96, 86, 45, 66, 45 and 120 per cent respectively compared to the control at 60 DAC. The days taken for flowering in P. indica-colonized plants were decreased by 12 days and the yield per plant in P. indica-colonized was 892.46 g against 449.51g. P.indica-colonization decreased incidence and severity of fungal and bacterial diseases under field conditions. Moreover, it recorded a drastic reduction in the severity of ToLCD by 58 percent. Tomato plants were subjected to pre- and postcolonization of P. indica challenged with ToLCV through wedge grafting at 2, 5, 10 and 15 days interval. Pre-colonized tomato plants decreased the severity of ToLCD by 66 per cent, while post-colonization decreased the severity by 26 per cent at various intervals of treatments. Further, P. indica-colonization promoted flowering and fruit set against complete flower abortion in non-colonized plants challenged with ToLCV under pot studies. The best treatments selected from pre- and post- inoculation of the fungus include P. indica-colonization followed by graft transmission of the virus after 15 days, and graft transmission of the virus followed by P. indica-colonization after 2 days respectively. Amplification of the virus by polymerase chain reaction (PCR) using Deng and AV/AC primers also revealed that the virus titer was the least in P. indica-pre-colonized plants that were inoculated with the virus after 45 DAC, followed by post-colonized plants that were inoculated with the virus. Nitro blue tetrazolium (NBT) for superoxides and diamino benzidine (DAB) for H2O2 staining recorded a reduction in ROS production in both pre- and post- P. indica-colonized tomato plants artificially inoculated with ToLCV against the virus alone. The decrease in ROS production and disease severity in the fungus colonized plants inoculated with the virus were attributed to the increased total soluble proteins and enhanced activities of antioxidant enzymes. The substantial increase in antioxidant enzymes viz., peroxidase, phosphatase, superoxide dismutase, catalase and glutathione reductase and glutamate synthase were observed in P. indica-colonized plants against the control; and for both pre- and post- P. indica-colonized plants artificially inoculated with ToLCV against the virus alone. Thus, the present study reveals that P. indica can be exploited for the management of ToLCV on prophylactic and curative bases with the best results in the former. P. indica colonized plants enhanced the over all growth and productivity; and drastically reduced the severity of ToLCD under both controlled and open field conditions.Item Elucidating the role of growth promoting endophytic fungus Piriformospora indica for water stress tolerance in rice (Oryza sativa L.)(Department of Plant Physiology, College of Agriculture ,Vellayani, 2022-03-10) Lekshmi, Mohan S; Beena, RThe research work titled “Elucidating the role of growth promoting endophytic fungus Piriformospora indica for water stress tolerance in rice (Oryza sativa L.)” carried out at Department of Plant Physiology, College of Agriculture, Vellayani during 2020-2021 with the objective to elucidate the changes in morphological, physiological, biochemical and molecular mechanisms associated with water stress tolerance in Piriformospora indica-colonised rice. The roots of Ptb 23, Ptb 24, Ptb 29 and Ptb 30 varieties’ seedlings were appraised microscopically for root colonisation after germinating in trays containing P. indica-infused composted coir pith- cow dung (1:1) mixture maintained in temperature and humidity-controlled conditions. Isolated or chains of double walled pear shaped chlamydospores were observed in the root sections of seedlings in trays with P. indica after seven days of cocultivation. On treatment with Poly Ethylene Glycol (PEG) 6000 at concentrations of 0, 5,10, 15, and 20 per cent, the varieties on root colonisation with P. indica showed higher water stress tolerance while the water stress tolerant varieties Ptb 29 and Ptb 30 showed tolerance to water stress even in the absence of P. indica. Shoot length, shoot and root dry weight, number of root branches, SVI 1 and SVI 2 increased in P. indica-colonised plants than non-colonised plants under water stressed condition, while the root length decreased. Based on the observations on different parameters, Ptb 29 was considered as the best water stress tolerant variety and as Ptb 23 performed better than Ptb 24 under water stress condition, the former was selected as the best water-stress susceptible variety. The P. indica-colonised and non-colonised rice seedlings of Ptb 23 and Ptb 29 were the evaluated for water stress tolerance during the different growth stages by studying the morphological, physiological, biochemical, yield parameters and molecular aspects. P. indica-colonised plants under water stress exhibited higher shoot and root length, shoot and root dry weight, root-shoot ratio and root volume, however, it was more conspicuous in the water stress susceptible variety. The enhanced plant shoot-root growth and biomass production as well as other changes in plant morphology after P. indica-colonisation can be related to the plant response to increase auxin level in roots either produced by the fungus or by the plant due to stimulation by the endophyte. Relative water content was found to be higher in water stressed P. indica-colonised plants in both Ptb 23 and Ptb 29, although the effect of colonisation was more visible in Ptb 23. There was significant increase in the specific leaf area in P. indica-colonised plants of both varieties than non-colonised plants. In the current study, rice plants of Ptb 29 under water stressed condition had higher cell membrane stability index than Ptb 23. Plants in the presence of P. indica showed significantly reduced MDA content which indicates that P. indica lowers the adverse consequences of drought. Ptb 29 had lower MDA content than Ptb 23, which further established that Ptb 29 is more tolerant to water stress. Gas exchange parameters like stomatal conductance, transpiration rate and photosynthetic rate showed increase in P. indica-colonised plants than in non-colonsed plants. Retention of chlorophyll was significantly higher in water stressed plants of Ptb 29 than Ptb 23. The increased activity of antioxidant enzymes such as superoxide dismutase and catalase in P. indica-colonised plants during stressed condition reduced ROS levels in cells and thereby minimizing or preventing damage to cellular components. P. indica-colonised plants showed a reduced invertase activity than non-colonised plants. P. indica-colonisation improved the number of tillers, productive tillers as well the panicle length in plants under water stressed condition. The spikelet sterility was higher in non-colonised and water stressed plants. P. indica-colonisation contributed to a considerable increase in grain weight in Ptb 23. All these factors contributed to the final yield of the plant and unsurprisingly, P. indica-colonised plant sets produced more yield than non-colonised plant sets under water stressed conditions. Even though Ptb 29 produced more yield., P. indica-colonisation was more noticeable and effective in the water stress susceptible variety- Ptb 23. Keeping in view of our results, it can be emphasised that P. indica can mitigate the ill effects of water stress. The findings obtained from this study can be used as a foundation for future lines of research related to rational improvement of rice plants against water stress using endophytes.Item Elucidating the role of growth promoting endophytic fungus Piriformospora indica for water stress tolerance in rice (Oryza sativa L.)(Department of Plant Physiology, College of Agriculture, Vellayani, 2022-10-03) Lekshmi Mohan S; Beena RThe research work titled “Elucidating the role of growth promoting endophytic fungus Piriformospora indica for water stress tolerance in rice (Oryza sativa L.)” carried out at Department of Plant Physiology, College of Agriculture, Vellayani during 2020-2021 with the objective to elucidate the changes in morphological, physiological, biochemical and molecular mechanisms associated with water stress tolerance in Piriformospora indica-colonised rice. The roots of Ptb 23, Ptb 24, Ptb 29 and Ptb 30 varieties’ seedlings were appraised microscopically for root colonisation after germinating in trays containing P. indica-infused composted coir pith- cow dung (1:1) mixture maintained in temperature and humidity-controlled conditions. Isolated or chains of double walled pear shaped chlamydospores were observed in the root sections of seedlings in trays with P. indica after seven days of cocultivation. On treatment with Poly Ethylene Glycol (PEG) 6000 at concentrations of 0, 5,10, 15, and 20 per cent, the varieties on root colonisation with P. indica showed higher water stress tolerance while the water stress tolerant varieties Ptb 29 and Ptb 30 showed tolerance to water stress even in the absence of P. indica. Shoot length, shoot and root dry weight, number of root branches, SVI 1 and SVI 2 increased in P. indica-colonised plants than non-colonised plants under water stressed condition, while the root length decreased. Based on the observations on different parameters, Ptb 29 was considered as the best water stress tolerant variety and as Ptb 23 performed better than Ptb 24 under water stress condition, the former was selected as the best water-stress susceptible variety. The P. indica-colonised and non-colonised rice seedlings of Ptb 23 and Ptb 29 were the evaluated for water stress tolerance during the different growth stages by studying the morphological, physiological, biochemical, yield parameters and molecular aspects. P. indica-colonised plants under water stress exhibited higher shoot and root length, shoot and root dry weight, root-shoot ratio and root volume, however, it was more conspicuous in the water stress susceptible variety. The enhanced plant shoot-root growth and biomass production as well as other changes in plant morphology after P. indica-colonisation can be related to the plant response to increase auxin level in roots either produced by the fungus or by the plant due to stimulation by the endophyte. Relative water content was found to be higher in water stressed P. indica-colonised plants in both Ptb 23 and Ptb 29, although the effect of colonisation was more visible in Ptb 23. There was significant increase in the specific leaf area in P. indica-colonised plants of both varieties than non-colonised plants. In the current study, rice plants of Ptb 29 under water stressed condition had higher cell membrane stability index than Ptb 23. Plants in the presence of P. indica showed significantly reduced MDA content which indicates that P. indica lowers the adverse consequences of drought. Ptb 29 had lower MDA content than Ptb 23, which further established that Ptb 29 is more tolerant to water stress. Gas exchange parameters like stomatal conductance, transpiration rate and photosynthetic rate showed increase in P. indica-colonised plants than in non-colonsed plants. Retention of chlorophyll was significantly higher in water stressed plants of Ptb 29 than Ptb 23. The increased activity of antioxidant enzymes such as superoxide dismutase and catalase in P. indica-colonised plants during stressed condition reduced ROS levels in cells and thereby minimizing or preventing damage to cellular components. P. indica-colonised plants showed a reduced invertase activity than non-colonised plants. P. indica-colonisation improved the number of tillers, productive tillers as well the panicle length in plants under water stressed condition. The spikelet sterility was higher in non-colonised and water stressed plants. P. indica-colonisation contributed to a considerable increase in grain weight in Ptb 23. All these factors contributed to the final yield of the plant and unsurprisingly, P. indica-colonised plant sets produced more yield than non-colonised plant sets under water stressed conditions. Even though Ptb 29 produced more yield., P. indica-colonisation was more noticeable and effective in the water stress susceptible variety- Ptb 23. Keeping in view of our results, it can be emphasised that P. indica can mitigate the ill effects of water stress. The findings obtained from this study can be used as a foundation for future lines of research related to rational improvement of rice plants against water stress using endophytes.Item Prevalence of sesamum phyllody in Onattukara tract and evaluation of fungal root endophyte Piriformospora indica for its management(Department of Plant Pathology, College of Agriculture, Vellayani, 2022) Gifty, K J; KAU; Radhika, N SThe research work entitled ‘Prevalence of sesamum phyllody in Onattukara tract and evaluation of fungal root endophyte Piriformospora indica for its management’ was conducted during 2019-21 at Department of Plant Pathology, College of Agriculture, Vellayani and Onattukara Regional Agricultural Research Station with the objectives to study the symptomatology, molecular detection and characterization of phytoplasma inciting sesamum phyllody disease in AEU 3 (Onattukara tract); and evaluation of fungal root endophyte P. indica for its management. Phytoplasma infected sesamum samples were collected from D and F blocks of Onattukara Regional Agricultural Research Station and Karthikapally. Karthikapally recorded highest disease incidence (39.44 per cent) and vulnerability index (23.75). Chocolate weed, Melochia corchorifolia, was found to be exhibiting symptoms of shoot proliferation. Hoppers collected from the infected fields were identified as Orosius albicintus, Hishimonas phycitis and Nephotettix sp. Disease symptoms were observed at the stage of flowering of sesamum plants in all the sampled locations. The associated symptoms were reduction in internodal length of stem, axillary bud proliferation, thickening of the floral veins, phyllody and floral proliferation. Microtome sections of infected and healthy leaf, stem of sesamum stained with 4,6-diamidino-2-phenylindole (DAPI) stain, and observed under fluorescence microscope emitted diffuse fluorescence from the infected tissues, which was brighter than the one from the parenchymal cells indicating the presence of phytoplasma in the infected tissues. Studies on variations in the level of gibberellic acid (GA) and indole-3-acetic acid (IAA) in phyllody infected and healthy sesamum was undertaken. GA content was increased by 2.25 times and 10.46 times, and IAA content was decreased by 1.25 times and 1.97 times in leaves and flowers of infected samples compared to the healthy samples. Molecular characterization of sesamum phyllody was performed with leaf samples collected from ORARS lowland, ORARS upland, Vellayani and Karthikapally. Amplicons of 1.4kb was obtained by amplifying with universal primers P1/P6 for detection of phytoplasma. The sequences obtained were subjected to BLAST analysis and the 16S rDNA gene sequence showed that all the isolates shared more than 99 per cent similarity with that of the ‘Candidatus phytoplasma aurantifolia’ strains in GenBank data base. In the phylogenetic tree constructed, the sesamum phyllody phytoplasma under study clustered with the 16SrII group (Candidatus Phytoplasma aurantifolia) phytoplasmas causing sesamum phyllody in various regions. The virtual RFLP pattern generated by iPhyClassifier, derived from 16S rDNA fragment was found to be identical to the reference pattern of 16Sr group II, subgroup D (GenBank accession: Y10097). Based on the results obtained from sequence analysis and virtual RFLP pattern, the phytoplasma associated with sesamum phyllody was identified as ‘'Candidatus Phytoplasma aurantifolia”-related strain belonging to subgroup 16SrII-D. P. indica obtained from Department of Plant Pathology, College of Agriculture, Vellayani was mass multiplied in sterilized coir pith: FYM mixture (1:1) amended with 2 per cent gram flour and sesamum seeds were sown. Colonization was observed seven days after germination. Wedge grafting was standardized in sesamum at 30 days after germination. Pot culture experiment was conducted to evaluate the effect of P. indica against phytoplasma causing sesamum phyllody, by grafting the colonized and non-colonized plants with infected scion. P. indica colonization could significantly reduce the incidence and severity of infection. After 30 and 45 days of grafting, an incidence of 20 and 60 per cent, and severity of 5 and 50 were recorded in the colonized plants grafted with infected scion, whereas an incidence of 60 and 80 per cent and severity of 45 and 75 were recorded in non-colonized plants grafted with infected scion. In colonized plants, enhanced shoot and root length at 30 and 55 days after germination were recorded and also earliness in flowering compared to noncolonized plants. Hence the associated symptoms of phytoplasma infection in sesamum are virescence, thickening of floral veins, phyllody and floral proliferation. The study revealed the association of Candidatus phytoplasma aurantifolia group with sesamum phyllody prevalent in Onattukara tract. The evaluation of beneficial fungal root endophyte P. indica against phytoplasma revealed delayed expression of symptoms in the colonized plants.