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ThesisItem Open Access Evaluation of Piriformospora indica against Piper yellow mottle virus in Black pepper(Department of Plant Pathology, College of Agriculture, Vellayani, 2021) Lekshmi, R Krishnan; KAU; Joy, MThe study entitled “Evaluation of Piriformospora indica against Piper yellow mottle virus (PYMoV) in black pepper” was conducted in Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during 2019-2021, to evaluate the efficiency of the root colonizing endophytic fungus P. indica in managing PYMoV in black pepper, and to elucidate the role of Reactive Oxygen Species and antioxidant enzymes in the tripartite interaction. A survey conducted in the instructional farm of College of Agriculture, Vellayani, to identify and collect PYMoV-infected black pepper plants showed that the surveyed varieties viz., Panniyur 1 to 7 and Karimunda were moderately to highly susceptible to PYMoV infection, with the disease incidence ranging from 10 to 91 per cent and disease severity as vulnerability index from 30 to 58. PYMoV-infected black pepper plants showed symptoms like chlorotic flecking, mottling, light necrosis, crinkling and distortion of leaves, reduction in leaf size and stunted growth. Presence of the virus in the samples was confirmed by polymerase chain reaction (PCR) with oligonucleotide primers specific to open reading frame III (ORF III) of PYMoV. P. indica culture obtained from Department of Plant Pathology was maintained in potato dextrose agar (PDA) and potato dextrose broth (PDB) media. P. indica was mass multiplied in coirpith-dried farm yard manure (FYM) mixture (1:1) amended with two per cent gram flour and was allowed to colonize on rooted cuttings of black pepper. P. indica colonization enhanced different biometric characters at different intervals, compared to the non-colonized control plants. P. indica colonization resulted in 25 per cent more shoot length, 23 per cent more root length, 35 per cent more number of leaves, 23 per cent more leaf area and 27 per cent more number of primary roots than the control plants at 150 days after co-cultivation (DAC). A pot culture experiment was laid out to evaluate the efficiency of prophylactic as well as curative application of P. indica, in managing PYMoV of black pepper. P. indica colonization significantly reduced the incidence and severity of PYMoD, upon pre- as well as post- inoculation of the virus, compared to the control plants. Plants colonized with P. indica and later inoculated with PYMoV as well as plants infected by PYMoV and later colonized by P. indica, both recovered the loss in biomass caused by the virus infection, compared to the non-colonized plants challenged by the virus. However, prophylactic treatment with P. indica resulted in better control of the disease compared to the curative treatment. Amplification of the virus by PCR also revealed that the virus titer was the least in P. indica-colonized plants that were post-inoculated with the virus after 30 DAC, followed by pre-inoculated plants that were later colonized with P. indica. The virus titer was the highest in the non-colonized plants inoculated with the virus. In order to evaluate the performance of P. indica in modulating the biochemical responses of black pepper plants against PYMoV infection, the activity of various enzymes which are known to play a role in the antioxidant defense systems of plants viz. catalase, peroxidase, glutathione reducatse, glutamate synthase, phosphatase and superoxide dismutase were assayed. A significant increase in the activity of these antioxidant enzymes was observed in the P. indica-colonized plants compared to the control plants, upon pre- as well as post- inoculation of PYMoV. Thus, the better performance of P. indica-colonized plants upon PYMoV infection can be correlated to the increased activities of antioxidant enzymes. PYMoV-infected plants showed increased accumulation of reactive oxygen species like superoxide anions and hydrogen peroxide, which cause oxidative stress in the plants. P. indica-colonization was able to mitigate the increase in ROS concentration caused due to PYMoV infection, thus reducing the symptoms. Thus the study reveals that, co-cultivation of rooted cuttings of black pepper (obtained from disease-free mother plants) with P. indica and keeping them under insectproof conditions for around 30 days (for successful establishment of the fungus inside the root cortex) and then planting them in the main field is a reliable and chemical-free method to control PYMoD in black pepper. Bio-protective action of P. indica might be attributed to the increased activity of antioxidant enzymes, which in turn leads to suppression in ROS accumulation in the colonized plants, upon being challenged by the virus.ThesisItem Open Access Varietal screening and management of anthracnose of black pepper using new generation fungicides(Department of Plant Pathology, College of Agriculture, Vellayani, 2021) 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 defense mechanism(Department of Plant Pathology, College of Agriculture, Vellayani, 2021) Veni Krishna, K C; KAU; Dhanya, M KThesisItem Open Access Etiology and characterization of diseases of Anthurium (Anthurium andraeanum L.) in Kerala(Department of Plant Pathology, College of Agriculture, Vellanikkara, 2021) Nitha, Rafi; KAU; Anita Cherian, KAnthurium (Anthurium andraeanum L.), a native of Tropical America is a perennial herbaceous plant commercially grown for its attractive coloured spathe and green shiny foliage. These have great economic value in the global flower trade that led to the budding of many urban and rural anthurium entrepreneurs in the countryside, particularly in the state of Kerala. During the recent past, the cultivation of this high valued cut flower crop is challenged by many biotic factors and the changes in climate scenario occurred recently led to the emergence of various new pests and diseases. Hence the present study was undertaken to identify and document the diseases affecting anthurium grown in the state of Kerala and the characterization of associated pathogens. Purposive sampling surveys were conducted in the anthurium cultivated locations of six districts viz. Thrissur, Ernakulam, Palakkad, Malappuram, Kozhikode and Wayanad during the period from October, 2020 to July, 2021. The incidence and severity of various symptoms were assessed and collected the samples for further studies. Twelve leaf spots (KMALS, PBALS, VFNLS, VCNLS1, VCNLS2, IJKLS, KKYLS, OKMLS, OLRLS, TLRLS, TBMLS and NLBLS) two leaf blights (VFNLB and OLKLB), one root rot (VCNRR), one wilt (MNTLW) four inflorescence rots (MNTSR, CKDSR, PNMSR and ALVSR) and one mosaic symptom (VCNML) were collected during the survey. Among the leaf spots, PBALS recorded the highest per cent disease severity (PDS) of 58.23 followed by NLBLS (46.66 %) and ALVSR recorded the highest severity of 32 per cent among inflorescence rots. Isolation and pathogenicity studies of the associated pathogens yielded 18 fungal and two bacterial isolates. Symptomatology of these diseases were studied both under natural and artificial conditions. The fungal pathogens were characterized and identified by studying cultural and morphological characteristics. Based on these characters, leaf spot pathogens were identified as Colletotrichum gloeosporioides, Lasiodiplodia theobromae, Phoma sp., Phomopsis sp., Corynespora sp., Pestalotiopsis sp. and those causing inflorescence rot as Colletotrichum gloeosporioides and Phomopsis sp. and root rot pathogen as Phytopythium sp. and wilt pathogen as Fusarium sp. The bacterial isolates causing leaf blight were identified as Xanthomonas sp. based on cultural, morphological and biochemical characteristics. The viral like symptoms subjected to electron microscopy revealed the absence of any virus particles. PCR amplification of Internal Transcribed Spacer (ITS) region of the fungal genome followed by sequencing and in silico analysis confirmed that the pathogen associated with TBMLS as C. queenslandicum, VCNRR as Phytopythium vexans, IJKLS as Diaporthe phaseolarum, KKYLS and OKMLS as Phomopsis heveicola, NLBLS as Lasiodiplodia theobromae, TLRLS as Pseudopestalotiopsis thea, VCNLS2 and OLRLS as Corynespora cassiicola, MNTLW as Fusarium fujikuroi. The pathogens associated with KMALS, PBALS, VFNLS, MNTSR, PNMSR and ALVSR belong to Colletotrichum gloeosporioides species complex. The bacterial isolates (VFNLB and OLKLB) were confirmed as Xanthomonas axonopodis through the amplification of 16S rRNA region of the genome followed by sequencing and in silico analysis. An in vitro experiment was conducted to study the efficacy of fungicides and biocontrol agents/bioformulations against major fungal pathogens. Fungicides viz. propineb, difenoconazole, carbendazim 12 % + mancozeb 64 %, cymoxanil 8% + mancozeb 64 % and Bordeaux mixture were highly effective against L. theobromae and Colletotrichum sp. (PBALS) as it resulted in cent per cent inhibition of these two pathogens at all the three doses. In the case of P. vexans, fungicides viz. propineb, azoxystrobin, carbendazim 12 % + mancozeb 64 %, cymoxanil 8% + mancozeb 64 % and Bordeaux mixture were found to be effective even at a lower dose. Among the three chemicals tested against Xanthomonas axonopodis, maximum inhibition was recorded in the case of streptocycline. Dual culture and filter paper disc method revealed that the biocontrol agents/formulations viz. Trichoderma sp., PGPR-II and PGPM were effective against all the tested fungal and bacterial pathogens. Pseudomonas fluorescens was found effective against three fungal and the bacterial pathogen.
