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ThesisItem Open Access Host range studies and management of anthracnose of nutmeg caused by colletotrichum spp.(Department of Plant Pathology, College of Agriculture, Vellayani, 2020) Bommana, Divya; KAU; Heera, GThe study entitled “Host range studies and management of anthracnose of nutmeg caused by Colletotrichum spp .” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2018-2020 with the objectives to characterize the causal agent, study the host range of Colletotrichum spp. causing anthracnose and to develop effective management strategy to control the disease by using new generation fungicides. As a part of the study, anthracnose infected samples were collected from four nutmeg growing districts of Kerala viz., Thiruvananthapuram, Kottayam, Ernakulam and Idukki. For the infected sample collections, three locations from Thiruvananthapuram (Vellayani, Karamana and Palode), two locations from Kottayam (Kumarakom and Vaikom), six locations from Idukki (Myladumpara, Pampadumpara, Adimali, Kambilikandam, Panickankudy and Kattapana) and one location from Ernakulam (Kadungalloor) were surveyed. Disease incidence and severity were assessed from the surveyed locations. The highest disease incidence and severity were observed in Kadungalloor (DI - 90 % and PDI - 56.40 respectively) followed by Kumarakom (DI - 80 % and PDI - 41.33 respectively) and the lowest disease incidence and severity in Myladumpara (DI - 20 % and PDI - 15.53 respectively). The symptoms of the anthracnose on nutmeg appeared as small necrotic spots with a prominent yellow halo on the leaf lamina. Several lesions coalesced together resulted in leaf blight, shot hole and defoliation. In Kambilikandam and Panickankudy. fruit rot was also observed along with leaf spot. The cultures of Colletotrichum spp. were isolated from the infected samples from different locations. Eighteen pure cultures of Colletotrichum sp. (C1 to C18) were obtained. Seven isolates of Colletotrichum sp. were selected for further studies based on the days taken for symptom development and rate of lesion development. The pathogenicity of the seven isolates of Colletotrichum sp. from different locations were proved by Koch postulates. The morphological and culture characters of the seven different isolates were studied in potato dextrose agar (PDA) medium. The isolated cultures of Colletotrichum sp. produced whitish to greyish radiating mycelium; later turning to off white to pink coloured fluffy to sparse mycelium with regular margins. Days taken to grow the entire Petri dish ranged from 7 to 10. The mycelium of the fungus was hyaline and septate; and its width ranged from 0.46 μm to 2.48 μm. The conidia were single celled with an oil globule at the centre and wereoblong or dumbbell shaped. The conidial size varied from 7.87 to 19.97 μm x 3.26 to 5.68 μm. The isolates were morphologically identified as C. gloeosporioides. The pathogenic variability of the seven isolates of C. gloeosporioides was assessed on detached nutmeg twigs by virulence rating. The isolate C4 was identified as the most virulent isolate which produced lesion size of 11.89 cm and 16.81cm at 7 DAI and 9 DAI respectively. The isolate C4 produced symptoms within two days after artificial inoculation and had a higher rate of lesion development of 4.12 cm day -1 . The other isolates took 3 to 4 days for symptom appearance on artificial inoculation of the pathogen. Host range of the most virulent isolate of C. gloeosporioides (C4) obtained from nutmeg was studied in perennial tree spices viz., clove, cinnamon, all spice, betel vine, black pepper and coconut. C. gloeosporioides isolate of nutmeg is capable of infecting the above- mentioned host plants. The isolate produced symptoms in all the hosts within 2 to 4 DAI and the symptoms developed varied from brown lesions, brown lesions with a shot hole to necrotic spots with prominent yellow halo. The maximum lesion size of 2.43 cm was observed in clove and minimum lesion size of 1.31 cm in all spice. In vitro screening of new generation fungicides revealed that triazole group fungicide propiconazole 25EC at 100 ppm and combination fungicides, carbendazim 12% + mancozeb 63% at 25 ppm; and Trifloxystrobin 25% + Tebuconazole 55% WP at 100 ppm concentration were the most effective in completely inhibiting the mycelial growth of the pathogen. The present study revealed the wide host range of the C. gloeosporioides isolate of nutmeg and also the effectiveness of new generation fungicides in managing the pathogen. The future line of work should include molecular variability between various isolates, cross infectivity among the isolates in other perennial hosts, and the efficacy of new generation fungicides under field condition.ThesisItem Open Access Varietal screening and management of anthracnose of black pepper using new generation fungicides(Department of Plant Pathology, College of Agriculture, Vellayani, 2020) Athira, K; KAU; Heera, GThe study entitled “Varietal screening and management of anthracnose of black pepper using new generation fungicides” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2018 - 2020 with the objective to screen KAU varieties and most popular local cultivar Karimunda for resistance against anthracnose of black pepper caused by Colletotrichum gloeosporioides (Penz.) Penz. and Sacc. and evolve management strategy using new generation fungicides. As a part of the study, diseased samples were collected from three black pepper growing tracts of Kerala viz., Thiruvananthapuram, Wayanad and Idukki. Sample collections were made from two locations from Thiruvananthapuram (Kowdiar and Vellayani), Wayanad (Meenangadi and Ambalavayal) and Idukki (Myladumpara, Pampadumpara, Kattapana and Kambilikandam). Disease incidence and severity were assessed from the surveyed locations. The highest percentage disease index was observed in Myladumpara (50.28%) followed by Kattapana (48.62%). Weather parameter viz., temperature, relative humidity and rainfall were recorded during the survey period. The weather parameters viz., low temperature, high relative humidity and heavy rainfall favoured the incidence of anthracnose. The symptoms of the anthracnose appeared as small necrotic spots with a yellow halo on the leaf lamina. Several lesions coalesce together resulted in leaf blight and defoliation. In Pampadumpara, spike infection was also observed along with leaf spot. Colletotrichum cultures were isolated from the diseased sample by tissue isolation technique and eight pure cultures of Colletotrichum sp. (C1 to C8) were obtained. The pathogenicity of the eight isolates of Colletotrichum sp. from different locations were proved by Koch postulates. The morphological characters of the eight different isolates were studied in potato dextrose agar (PDA) medium. The isolated cultures of Colletotrichum sp. produced whitish with yellowish orange centre to light pink, off white to greyish coloured colony having fluffy, cottony to sparse mycelial growth with regular margins. Days taken to grow the entire petridish ranged from 7.25 to 9.75 days. The mycelium of the fungus was hyaline and septate, and its width ranged from 2.21 - 3.45 μm. The septal distance of the different Colletotrichum isolates ranged between 8.50 - 21.23 μm. The conidia were single celled with an oil globule at the centre. The conidial shape was either cylindrical, oblong or dumbbell. The conidial and appressorial size varied from 9.4 - 12.1 μm x 3.6 - 4.6 μm and 8.5 – 11.2 μm x 3.5 – 4.3 μm respectively. The isolates were identified as Colletotrichum gloeosporioides. The pathogenic variability of the eight C. gloeosporioides isolates were assessed on three black pepper varieties viz., Panniyur 1, Panniyur 3 and Karimunda by virulence rating. The isolate C7 was identified as the most virulent isolate which produced lesion size of 1.92 cm, 2.40 cm, and 3.22 cm on Panniyur 1, Panniyur 3 and Karimunda respectively at 5 days after inoculation (DAI). The isolate C7 produced symptoms within two days after artificial inoculation in the three varieties tested with a higher rate of lesion development of 0.40 (Panniyur 1), 0.49 (Panniyur 3) and 0.66 (Karimunda) cm day-1. KAU varieties (Panniyur 1 to 8) and local cultivar Karimunda were screened against the most virulent isolate of C. gloeosporioides. Among the varieties screened, Panniyur 4 was found to be highly susceptible with highest PDI of 51.43 (7 DAI), whereas Panniyur 2 had the lowest PDI of 14.28 (7DAI) followed by Panniyur 8 with PDI 20.00 % (7DAI) and were found to be tolerant to anthracnose infection. Panniyur 1, Panniyur 7 and Panniyur 5 were also found to be moderately susceptible. The pathogen produced symptoms in susceptible varieties within 2 DAI, whereas the tolerant varieties took 3-4 days to initiate the infection. In vitro screening of new generation fungicides revealed that kresoxim methyl of strobilurin and tebuconazole of triazole were the most effective in inhibiting mycelial growth of C. gloeosporioides (80.37% and cent percent respectively). The combination fungicide carbendazim 12% + mancozeb 63 % completely inhibited the mycelial growth at 25, 50 and 100 ppm. The combination fungicides azoxystrobin 11% + tebuconazole 18.3% SC and trifloxystrobin 25% + tebuconazole 55% WP were also effective against the pathogen at 100 ppm. The contact fungicide copper oxychloride was ineffective against the pathogen @ 10, 25, 50 and 100 ppm. The present study revealed the use of tolerant varieties along with need based application of new generation fungicides to keep the destructive disease under control. The future line of work should include screening of more black pepper varieties under field condition to assess their reaction to anthracnose, elucidation of the factors governing resistance to the disease and the efficacy of new generation fungicides under field condition.ThesisItem Open Access Eco-friendly management of Fusarium rot in cardamom and its impact on soil health and plant defence mechanism(Department of Plant Pathology, College of Agriculture, Vellayani, 2020) Veni Krishna, K C; KAU; Dhanya, M KThe study entitled ‘Eco-friendly management of Fusarium rot in cardamom and its impact on soil health and plant defense mechanism’ was conducted at College of Agriculture, Vellayani and Cardamom Research Station Pampadumpara during 2018- 2020 with the objective to assess Fusarium rot severity and pathogen variability in Idukki district, develop an effective ecofriendly management practices for the disease and study the impact of the practices on the soil and plant health. Six isolates of Fusarium oxysporum Schlecht were obtained, three each from infected pseudostem and roots collected from Kattapana and Nedumkandam blocks of Idukki district. The isolate, Fp1 from pseudostem (Pampadumpara panchayath) was identified as most virulent based on virulence rating (viz., days taken for symptom development, lesion length, mycelial growth), and was used for further studies. Inoculation of the root isolate (Fr1) also produced typical lesion on pseudostem indicating that the, fungus can also attack pseudostem besides the roots. But the root isolate took more days (40 days) for symptom development compared to pseudostem isolate (29 days). The morphological characters including the colour, colony characters, hyphal and conidial characters as well as rate of growth in Petri dish confirmed the fungus as F. oxysporum. In vitro studies revealed that the isolates from pseudostem and roots showed significant variation in their morphological, cultural and pathological characters. A pot culture experiment was conducted to assess the efficacy of selected bioagents (individually and in combination) for the management of the disease at CRS Pampadumpara in CRD using nine treatments with three replications. Pathogen inoculum (150 gm/10 kg soil) multiplied in sand-maize flour medium was standardised as the inoculum level enough for cent per infection on the pseudostem resulting in complete crop loss. Soil application of vermiculite based AMF inoculum (20 g) with @ 2% Pseudomonas fluorescens (1 L/10 kg soil) per 10 kg soil at the time of planting along with 2% P fluorescens spray @ 0.5 L/ plant at monthly interval for three times resulted in effective disease management (disease incidence: 40% and disease severity: 24.26%) compared to the inoculated control (disease incidence: 100% and disease severity: 69.38 %). Studies on the population dynamics of the pathogen and the biocontrol agents at periodical interval upto three months revealed significant reduction in the pathogen antagonist ratio and enhancement in AMF colonization of the treatment plants. Among the best treatment the combination of AMF and P. fluorescens resulted in good biometric characters of the treatment plants (plant height: 96.50 cm ,leaf length: 62.50 cm and number of leaves: 15.75) compared to control (plant height: 41 cm ,leaf length: 30 cm and number of leaves: 7.50) through enhancement of soil nutrients (P, K, Ca and Mg) and plant nutrient status (K, Mg, S and B) compared to control plants. Laboratory studies also revealed the induction of defense related enzymes (phenol, ortho dihydroxy phenol, peroxidase, polyphenol oxidase, and β 1,3 glucanase) in high concentration as a response to the application of above treatments. Thus, the present study revealed that Fusarium rot of cardamom a devastating disease can be effectively managed by soil application of vermiculate based AMF inoculum (20 g) with 2 per cent P. fluorescens (1L/10 kg soil) per 10 kg soil at the time of planting along with 2 per cent P. fluorescens spray at 0.5 L per plant at monthly interval for three times; this treatment also resulted in good vegetative growth of cardamom plants therefore this can be used as an ecofriendly management strategy for the production of good quality cardamom.ThesisItem Open Access Strain improvement of Trichoderma spp. by protoplast fusion(Department of Plant Patholgy, College of Agriculture, Vellayani, 2020) Anit, Cyriac; KAU; Sible George, VargheseA study on “Strain improvement of Trichoderma spp. by protoplast fusion” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during the year 2018-2020, with the objective of improving the screened strains of Trichoderma spp. by protoplast fusion for spp. by protoplast fusion for increasing the antagonistic ability and related traits against soil borne pathogens. A survey was conducted in five agro-climatic zones of Kerala viz., Northern Zone, Central Zone, High Range Zone, Problem Area Zone and Southern Zone for collection of soil samples especially from forest soils. The collected soil samples were assessed for the population of Trichoderma spp. A total of 31 Trichoderma spp. isolates were obtained from the soil samples collected from five agro-climatic zones. Majority of the isolates were obtained from soils with pH of 6 to 7. Isolate TRMW2, TREN1, TREZ1, TREZ2, TREZ3, TRRN1, TRRN2, TRKR1, TRPN3, TRPN7, TRPN10 and TRPN18 exhibited full growth at four days after inoculation (DAI). The isolated Trichoderma spp. differed in growth rate and colony characters like colour of mycelium, texture of colony and sporulation pattern. Isolates of Trichoderma spp. from different zones exhibited in vitro inhibition against soil borne pathogens such as Pythium aphanidermatum and Rhizoctonia solani. Majority of the isolates displayed high inhibition per cent compared to KAU strain of Trichoderma sp. TRRN1, TRRN2, TRPN3, TRPN7, TRPN11, TRPN15 and TRKR2 isolates exhibited complete inhibition of P. aphanidermatum in dual culture experiment; whereas TREN1, TRMW2, TREZ1, TREZ2, TRKM1, TRPN7, TRPN9, TRPN14, TRPN15, TRPN17, TRPN18 and TRKR2 isolates exhibited complete inhibition of R. solani. Trichoderma isolates such as TRPN7, TRPN15 and TRKR2 exhibited complete inhibition against both the pathogens. Antagonistic properties viz., antibiosis, lysis and overgrowth of Trichoderma isolates against P. aphanidermatum and R. solani were observed. During the antagonist-pathogen interaction, isolates TRSN1, TRSN2, TRPN10, TRPN14, TRPN15, TRPN17 and TRPN18 exhibited high levels of antibiosis. Most of the isolates caused lysis of mycelium of the pathogens which resulted in formation of clear zones in dual culture. Overgrowth of the antagonist was another prominent antagonistic property observed in the majority of the isolates. Based on the antagonistic properties, the Trichoderma isolates viz., TRSN1, TRMW2 and TRPN14 were selected for the protoplast fusion. During the protoplast isolation, the maximum number of protoplasts was obtained after 2 h of incubation of mycelia of parental isolates with the lytic enzyme. Protoplast fusion was carried out between the selected isolates (TRSN1 x TRPN14, TRSN1 x TRMW2, and TRPN14 x TRMW2) in the presence of poly ethylene glycol (PEG 6000). Three protoplast fusants were selected using carbendazim-amended PDA medium. The protoplast fusants displayed fast growth on PDA medium and completely covered the Petri dish at 5th of growth. The colony characters of fusants varied from light to dark green mycelium with fluffy growth and scattered to circular green heavy sporulation. In vitro screening of protoplast fusants against P. aphanidermatum and R. solani revealed that highest inhibition against P. aphanidermatum was observed with fusant 3 (84.4%) followed by fusant 2 (74.44%). Highest inhibition against R. solani was observed with fusant 2 (100%) followed by fusant 3 (70.30%). Antagonistic properties viz., antibiosis, lysis and overgrowth were observed in the protoplast fusants. Among the three protoplast fusants, fusant 1 exhibited all the antagonistic properties against both the pathogens with heavy sporulation. Thus, the present study has thrown light in understanding the potential of protoplast fusion in evolving improved strains of Trichoderma spp. Protoplast fusion enhanced sporulation in fusants compared to the parents. Further studies need to be conducted for the biochemical and molecular characterisation of parental isolates and fusants. The parents and protoplast fusants also have to be evaluated for their in vivo efficacy against soil borne pathogens in major crops of Kerala.ThesisItem Open Access Characterization of fungal pathogen associated with leaf rot disease of coconut (Cocos nucifere L.) and In Vitro evaluation of phylloplane microflora as biocontrol agents(Department of Plant Pathology, College of Agriculture, Vellayani, 2020) Deena, Sebastian; KAU; Radhakrishnan, N VLeaf rot disease (LRD) is a major foliar disease affecting coconut plantations of Southern Kerala especially in root (wilt) affected areas. In this context, the study entitled ‘Characterization of fungal pathogen associated with leaf rot disease of coconut (Cocos nucifera L.) and in vitro evaluation of phylloplane microflora as biocontrol agents’ was conducted in the Department of Plant Pathology, College of Agriculture, Vellayani during the year 2018-2020, with the objective to identify and characterize the major fungal pathogens associated with the LRD of coconut and in vitro evaluation of phylloplane microflora of coconut against the pathogens. The isolation of LRD pathogens was carried out from six taluks of Thiruvananthpuram district such as Thiruvananthapuram, Neyyattinkara, Nedumangad, Chirayinkeezhu, Kattakada and Varkala. Three locations were selected from each taluk and a total of eighteen samples were collected during the study. The results revealed that the disease in Thiruvananthapuram district was caused by a spectrum of pathogens such as Colletotrichum gloeosporioides, Fusarium spp., Gliocladium sp., and Scytalidium sp. The LRD was caused either by a single pathogen or by combinations of pathogens. C. gloeosporioides and Fusarium spp. were found as the major pathogens of LRD based on the frequency of isolation. Each and every isolate of the same pathogen differed from one another in cultural characters and virulence. All the pathogens produced water soaked brown lesion on artificial inoculation on detached spindle leaves; though the days taken for symptom initiation and size of the lesion developed varied. The isolate C3 (Isolate from Anayara, Thiruvananthapuram taluk) was found to be more virulent among the C. gloeosporioides isolates; and among the Fusarium spp. isolates, the isolate F5 (Isolate from Alamkode, Chirayinkeezhu taluk) was found to be more virulent. By observing the spore characters of the isolates, it was found that the spore size and pigmentation of the culture haven’t any significance to the virulence of the pathogen. Dual inoculation of the major pathogens on detached spindle leaves caused severe incidence of the disease compared to the individual inoculation of the pathogens. This result indicated that the LRD caused by fungal complex is more severe than that caused by individual fungal isolates. There are phylloplane fungi existing on healthy leaves of the infected palm with enough inhibition potential to LRD. The phylloplane fungal isolate PF5 showed more per cent inhibition to mycelial growth of C. gloeosporioides (54.44%) followed by the isolate PF4 (43.33%); and the isolate PF4 showed more inhibition to Fusarium sp. (64.44%) followed by the isolate PF5 (45.55%) in the dual culture assay. The detached spindle leaf assay also supported the same fact that the isolate PF5 was observed to be more suppressive to the disease caused by C. gloeosporioides (28.77%) and the isolate PF4 was reported to have more suppression to the disease caused by Fusarium sp. (34.56%). These pre-treatment effects are more promising than Pseudomonas fluorescens PN026, but inferior to copper oxy chloride (0.2%). Thus, the present study revealed that the LRD of coconut in Thiruvananthapuram district is caused by a combination of pathogenic fungi viz., C. gloeosporioides, Fusarium spp., Gliocladium sp., and Scytalidium sp. Prophylactic application of the phylloplane fungal isolates PF4 and PF5 could reduce the LRD severity in vitro to a promising level and these isolate can be further tested for in vivo biocontrol potential before going for the development of a formulated product.ThesisItem Open Access Management of stem rot and foliar blight of cowpea (Vigna unguiculata (L.) Walp.)(Department of Plant Pathology, College of Agriculture, Padannakkad, 2020) Nayana Sunil, M V.; KAU; Susha S, TharaABSTRACT Management of stem rot and foliar blight of cowpea (Vigna unguiculata (L.) Walp.) Cowpea (Vigna unguiculata (L.) Walp.) is one of the highly demanding, nutritionally rich vegetable and legume crops cultivated in Kerala throughout the year. Stem rot and foliar blight disease in cowpea was emerged as a serious disease in farmer’s fields especially during monsoon period and resulted in severe yield reduction and economic loss. Hence present study was undertaken during 2018-2020 with an aim of identifying and characterizing fungal pathogen causing stem rot and foliar blight disease in cowpea and to evaluate biocontrol agents and chemical fungicides against it. Cowpea plants showing symptoms of stem rot and foliar blight disease were collected from six locations of Kasargod district such as Cheemeni, Pallikkara, Nileswar, Trikaripur, Udinur and Periya showing disease incidence ranging from 12 to 80 % and percent disease index of 8 to 51 %. Highest disease incidence recorded at Nileswar and lowest at Periya. Six isolates obtained were named with index ‘Sr’ such as Sr1, Sr2, Sr3, Sr4, Sr5 and Sr6. Pathogenicity test of six isolates were done in cowpea by soil inoculation and leaf inoculation methods. Isolate Sr3 produced symptom on stem at 2nd day and on leaf at 24 h of inoculation which was earlier than other isolates. Area of water soaked lesion on stem (6.4 cm2) and leaf (9.6 cm2) was also highest for Sr3 and observed fastest radial growth compared to others and covering 90 mm petridish at third day of inoculation. Based on disease development and growth rate on PDA, Sr3 was considered as most virulent isolate. Pathogen was identified based on cultural, morphological and molecular characterestics. Cottony and fluffy pure white colony with light zonation and presence of two types of fungal hyphae with clamp connection were observed. Two types of sclerotial bodies were detected and cross section of sclerotium revealed the presence of different layers such as outer thick skin, rind, cortex and medullary cells under compound microscope. Molecular identification based on D1/D2 region of LSU revealed 99.61 % similarity with Athelia rolfsii. Based on all these characteristics isolate Sr3 was identified as Sclerotium rolfsii. Symptoms associated with stem rot disease were water soaked lesion at the basal region of stem followed by wilting, yellowing of the aerial parts, necrosis, girdling and rotting of the stem. White mycelia and sclerotial bodies were formed at the infected portion. Foliar blight disease showed water soaked lesion on the leaf with concentric ring formation. Both mycelia and sclerotial bodies were also produced over the lesion. Effect of biocontrol agents on S. rolfsii tested by dual culture method explained higher inhibition per cent of Trichoderma harzianum followed by Trichoderma viride. Bacterial biocontrol agents showed least potential of antagonism in which Pseudomonas fluorescens exhibited zero inhibition on pathogen. Among the fungicides tested against S. rolfsii by poisoned food method, mancozeb 75WP (0.1 %, 0.2 % & 0.3 %) and propiconazole 25EC (0.05 %, 0.1 % & 0.2 %) were found best having 100 % inhibition over the pathogen whereas copper oxychloride 50 WP (0.1 %, 0.2 % & 0.3 %) and carbendazim 50WP (0.05 %, 0.1% & 0.2 %) were not effective. Compatibility of mancozeb, propiconazole and chlorothalonil were tested with T. harzianum and T. viride. Mancozeb (0.2 %) exhibited 100 % compatibility with T. harzianum and 92.39 % with T. viride. Propiconazole (0.1 %) was highly (100 %) incompatible with both of these followed by chlorothalonil (0.2 %). Field evaluation with most efficient fungicides, biocontrol agents and combination of both were experimented on cowpea (variety: Kanakamani) in pot culture method. Pre-sowing drenching with post sowing drenching and spraying of mancozeb (0.1 %) and propiconazole (0.05 %) at 20, 40, 60 days of sowing were found best for the management of stem rot and foliar blight disease having BC ratio of 1.9 followed by combinations of mancozeb and Trichoderma sp. Among the biocontrol agents T. harzianum were found superior than T. viride. Hence soil drenching at 7 days before sowing and soil drenching and aerial spraying at 20, 40, 60 days after sowing with mancozeb (0.1 %) or propiconazole (0.05 %) or mancozeb-Trichoderma combinations or T. harzianum (2 %) can be recommended for management of stem rot and foliar blight disease in cowpea. future line of work should be focused on field level study of stem rot and foliar blight disease in other districts of Kerala and to evolve local specific management with native isolates of biocontrol agents and their metabolites.ThesisItem Open Access Alginate based encapsulation of Pseudomonas fluorescens for management of soil borne pathogens(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2020) Sivadharshnapriya, R; KAU; Reshmy, VijayaraghavanBiological control, an eco-friendly and cost effective approach for plant disease management in agriculture has been practiced for several decades. Pseudomonas fluorescens, one such biocontrol agent is used to combat many phytopathogens. For commercial use, microbial inoculum should be supported by an appropriate formulation preventing a rapid decline of introduced microorganisms and extending their shelf-life. Various formulations available in the market are powder, liquid and granular formulations where such carrier based inoculants which generally faces problems like poor shelf life, high chances of contamination, bulk sterilization problem, unpredictable field performance and sometimes unavailability of good carrier materials. In order to overcome the disadvantages of these formulations, microencapsulation is one such alternate viable option prepared by using sodium alginate as a polymer which results in extended shelf-life and controlled microbial release from the formulation thus, enhancing their application efficacy. Hence, the present study was undertaken in the Department of Plant Pathology, College of Horticulture, Vellanikkara to develop alginate based formulation of Pseudomonas fluorescens for the management of soil borne pathogens. P. fluorescens, the reference culture of KAU was used for preparation of alginate beads. To improve the shelf life of alginate beads, King’s B broth was enriched with adjuvants viz., sugar source (mannitol and trehalose), wetting agent (PVP and PEG), adhesive (CMC and liquid paraffin) and surfactant (tween-80) in nine different treatments and was evaluated at monthly intervals. After nine months of observation, maximum population of P. fluorescens was recorded in treatments T1 (mannitol +PVP +CMC +tween-80) (1.33 x 108 cfu ml-1) and T3 (mannitol +PEG +CMC +tween-80) (1.66 x 108 cfu ml-1) compared to control which were selected for the preparation of beads. The beads were prepared from the above selected treatments as per the protocol of Bashan et al. (2002) with modifications. The beads were prepared in three different batches viz., beads from alginate alone, beads from alginate amended with skim milk and beads after secondary multiplication. Various parameters were standardised in order to prepare effective bead formulation. Beads from both alginate and alginate amended skim milk formulations produced from higher concentration of sodium alginate (3%) and calcium chloride (3.5% and 3.0%) solution with 60 min of curing time and 9 to 15 cm of falling distance produced perfectly spherical beads with maximum diameter of above 1.70 mm, higher bead weight of above 16.6 mg with more than 60 per cent bead yield. Such beads showed reduced swelling percentage which holds higher per cent of water content inside beads and lowest shrinkage percentage that facilitates higher survival and slow release of the bioagent for a longer period of time. Shelf life of P. fluorescens encapsulated in alginate beads alone prepared from two best treatments T1 (mannitol + PVP + CMC + tween-80) and T3 (mannitol + PEG + CMC + tween-80) showed a higher shelf life compared to alginate amended skim milk beads and beads after secondary multiplication. Higher bacterial entrapment were observed in alginate beads prepared from sodium alginate (3%) and calcium chloride (3.5% and 3.0%) respectively in treatments T3A12 (10.33 x 1020 cfu g-1) followed by treatments T3A11 (8.33 x 1020 cfu g-1), T1A6 (6.33 x 1020 cfu g-1) and T1A5 (5 x 1020 cfu g-1) respectively after four months of preparation. The alginate bead combinations B-1: T1 (mannitol + PVP + CMC + tween - 80) + sodium alginate (3%) + CaCl2 (3%) and B-2: T3 (mannitol + PEG + CMC + tween -80) + sodium alginate (3%) + CaCl2 (3%) were selected for in vitro evaluation studies against major soil borne pathogens and it was noticed that these formulations inhibited soil borne pathogens viz., Pythium aphanidermatum (100%) followed by Phytophthora nicotiana (72.22 and 77.77%) Ralstonia solanacearum (70.36 and 74.07%) and to some extent inhibition of Fusarium oxysporum (27.77 and 30.55%). However, no inhibition was observed on the growth of Sclerotium rolfsii and Rhizoctonia solani under in vitro conditions. Hence the study has clear by demonstrate a protocol to produce microbeads of P. fluorescens which are less bulky, non-toxic, biodegradable and enables slow and controlled release of the biocontrol agent and thus could maintain a bacterial population for a relatively longer period.ThesisItem Open Access Etiology and management of fruit rot of brinjal (Solanum melongena L.)(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2020) Aswathy, Peethambaran; Sainamol Kurian, PBrinjal belonging to the family Solanaceae is of great importance in human nutrition and is considered as the King of Vegetables. Despite being a versatile crop adapted to various agroclimatic regions and seasons, brinjal is affected by many diseases like Phomopsis blight and fruit rot, little leaf, damping off and bacterial wilt. In addition to these, recently a fruit rot caused by Phytophthora nicotianae is also reported from various parts of India (Jain et al., 1982 and Chowdappa et al., 2015). A preliminary study of the same has been conducted by Jose and Beena (2016). Considering the importance of Phytophthora spp. in the agricultural scenario of Kerala, the present investigation was carried out with the objectives to study the symptomatology and etiology of fruit rot of brinjal, screening popular brinjal varieties for resistance and to develop management strategy for the disease. Purposive sampling survey was conducted in different locations of Thrissur and Palakkad districts during June to October 2019, infected brinjal fruits were collected and symptoms were recorded. The incidence and severity of the disease in different locations ranged from 10 to 84 and 10 to 66 per cent respectively. Presence of a white mycelial growth was consistently observed on the infected fruits which on examination under microscope showed hyaline, coenocytic hyphae and pear shaped sporangia with papillae. The microorganism was isolated on carrot agar and pathogenicity was tested. Symptoms produced on artificial inoculation were also recorded. A total of four isolates of the pathogen were collected, purified and maintained for further studies. The isolates were named after the place of collection as VKA Ⅰ, VKA Ⅱ, KPD and NMA. Cultural and morphological characters of the isolates were studied on three different media viz., carrot agar, potato dextrose agar and coconut water agar. Colony characters like growth rate, colour, shape, texture of colony and sporulation varied among the isolates and media. All the four isolates produced coenocytic, hyaline, branched mycelium and thick walled, hyaline, globose chlamydospores. Non caducous, papillate, ovoid and obturbinate sporangia were borne terminal or intercalary on the sporangiophore in a simple sympodial fashion. Variation in dimensions of sporangia was observed among the isolates grown on different media. Based on morphology, all the isolates were identified as Phytophthora sp. Oospore production was not noticed on pairing of isolates suggesting that these belong to single mating type and express the typical heterothallic behaviour of the genus. Molecular characterisation of the four isolates revealed maximum similarity to P. nicotianae and phylogenetic analysis showed that isolates VKA Ⅰ, VKA Ⅱ and NMA are more closely related than KPD. Host range studies proved that the pathogen can infect other crops like tomato, chilli, bhindi, bitter gourd, arecanut, coconut, nutmeg, cocoa, papaya, rubber and black pepper on artificial inoculation. The most popular varieties of brinjal, released from Kerala Agricultural Universiy viz., Surya and Haritha were found to be susceptible to the pathogen. The results of in vitro evaluation showed that cymoxanil 8%+ mancozeb 64%, metalaxyl+ mancozeb, mancozeb, copper hydroxide, fosetyl- Al at 0.05 per cent concentration and Bordeaux mixture (0.5%) recorded cent per cent inhibition of the pathogen. However, azoxystrobin (0.05%) recorded 37 per cent inhibition only. Similarly Trichoderma sp. and Pseudomonas fluorescens recorded cent per cent and 69.40 per cent inhibition respectively. The leaf extracts of Capsicum frutescens and Boerhavia diffusa also gave significant inhibition of pathogen. In bioassay on detached fruits the highest disease reduction was recorded by mancozeb (72.38%) followed by cymoxanil 8%+ mancozeb 64% (66.26%). However biocontrol agent Trichoderma sp. also recorded 51.67 per cent reduction over control which was on par with fosetyl- Al, copper hydroxide and azoxystrobin. Leaf extract of Capsicum frutescens also reduced the disease by 39.18 per cent. Phytophthora fruit rot is an emerging disease and if left uncontrolled it will be a serious threat to brinjal cultivation. The study has confirmed the etiology and symptomatology of the disease and the wide host range of the pathogen. It was also proved that the four isolates of P. nicotianae collected in the study are confined to same mating type. The investigation has also revealed the efficacy of lower doses of commonly used fungicides, against P. nicotianae. Results of the study also suggested the potential of biocontrol agents and botanicals against the pathogen.ThesisItem Open Access Management of bhendi yellow vein mosaic virus using beneficial fungal root endophyte Piriformospora indica(Department of Plant Pathology, College of Agriculture, Vellayani, 2020) Chippy.; Joy, MThe study entitled ‘Management of Bhendi yellow vein mosaic virus using beneficial fungal root endophyte Piriformospora indica’ was conducted at College of Agriculture, Vellayani during 2018 - 2020 with the objective to evaluate beneficial fungal root endophyte, P. indica for the management of Bhendi yellow vein mosaic virus (BYVMV) and to elucidate the role of antioxidants in the tripartite interaction. BYVMV infected okra samples were collected from okra fields of 3 different locations of Thiruvananthapuram district viz., Vellayani, Pappanchani and Palappur. All the surveyed fields were severely infected and the highest disease incidence (D. I.) of 100% and vulnerability index (V. I.) of 71.07 was recorded in okra var. Kiran in Palappur. Characteristic symptoms observed in the surveyed locations include vein clearing, vein thickening, reduction in leaf size, stunted growth and reduced fruit set, fruits if formed were bleached, hardened and deformed. Vein clearing on flower buds was also observed. Total DNA of infected plant samples were isolated using Qiagen’s DNeasy Plant DNA extraction Mini Kit. The quantity and quality of DNA was assessed. The presence of the virus in the diseased samples were confirmed at molecular level by PCR using Deng and AV/AC primers specific to coat protein (CP) of BYVMV (Begomovirus) that could produce amplicons of 500 bp and 550 bp respectively in all infected samples. The pollen collected from the infected plants also produced amplicons confirming the presence of the virus. In vitro co-cultivation studies of P. indica in okra seedlings var. Salkeerthi in PNM medium revealed the fungal colonisation and formation of chlamydospores 25 days after co-cultivation. P. indica-colonisation enhanced shoot and root biomass; and produced more number of secondary roots. A pot culture experiment was conducted to evaluate the effect of P. indica-priming okra var. Salkeerthi under natural incidence of BYVMV. P. indica-priming significantly reduced the D. I. by 57 and V. I. by 56 per cent over control and also enhanced plant growth with increased root and shoot biomass, leaf area and plant height. There was 48 and 62 per cent increase in root and shoot fresh weight respectively in P. indica-colonised plants over control. Biochemical analysis of P. indica-primed and non-primed plants under natural incidence of BYVMV revealed increase in chlorophyll and total soluble proteins, and high activities of antioxidant enzymes in P. indica colonised plants. Total chlorophyll content was highest (1.32 mg g-1 fw) in P. indica-primed plants and least in BYVMV infected plants (0.20 mg g-1 fw). There was a 3-fold increase in total chlorophyll content in P. indica-primed plants under natural incidence compared to the diseased control. Chlorophyll b content was drastically reduced in diseased plants but P. indica-priming resulted in 3.2-fold increase in chlorophyll b. Total soluble protein content was increased by 121 per cent in P. indica-primed plants. Peroxidase (PO) and polyphenol oxidase (PPO) activities were increased by 94 per cent and 226 per cent respectively. There was 48 per cent increase in activities of both catalase and ascorbic acid oxidase in P. indica -primed plants over the diseased control. The decreased disease incidence and severity are attributed to the increased activities of antioxidant enzymes viz., peroxidase, polyphenol-oxidase, catalase and ascorbic acid oxidase. Effect of P. indica-priming (pre- and post- inoculation) on artificial inoculation of the virus by grafting was also evaluated. Both pre- and post-inoculation of the fungus in artificially infected okra plants reduced the V. I. by 56 and 24 per cent respectively. Similar trend as in case of natural incidence was also observed in biochemical activities in P. indica-primed okra plants under artificial inoculation. In pre-inoculation of P. indica followed by graft transmission of the virus, total chlorophyll and soluble protein contents were increased by 176 and 147 per cent respectively; PO and PPO activities by 109 and 176 per cent respectively; and catalase and ascorbic acid oxidase by 96 and 56 per cent respectively over the diseased control. Phosphatase activity was drastically decreased in the virus infected plants. The biochemical activities were significantly increased in post-inoculation of the fungus, but were not substantially as in the pre-inoculation followed by grafting of the virus. Thus, the present study revealed that P. indica could be exploited for the management of BYVMV on a prophylactic basis. Remission of the symptom in P. indica-colonised okra plants was attributed to the enhanced chlorophyll, soluble proteins, PO, PPO, catalase and ascorbic acid oxidase. Further studies should be conducted to elucidate the role of other antioxidants, and molecular mechanisms involved in this host-endophyte-virus interactions. Field and multi-locational studies should also be conducted for confirmation of the results.
