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ThesisItem Open Access Etiology and management of mosaic disease in ginger (Zingiber officinale Roscoe)(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Ananthu, N; KAU; Umamaheswaran, KThe study entitled ‘Etiology and management of mosaic disease in ginger (Zingiber officinale Roscoe)’ was conducted at the Department of Plant Pathology, College of Agriculture, Vellayani during 2015-2018 with the objectives to identify, characterize and sequence the genes of Ginger mosaic virus infecting ginger along with the management of the disease. As part of the study, the symptoms produced by the virus in ginger plants collected from the field and grown in glass house were observed. The symptoms on the leaves appeared as small light green flecks. These flecks eventually increased in size and formed streaks. The streaks were arranged parallel to the veins. The appearance of too many streaks on the leaves led to severe chlorosis and the leaves showed necrotic symptoms in the advanced stage. Transmission of the virus was tested in rhizome (seed material collected from infected plants) and mechanical inoculation was done using infected leaf sap to the healthy plants. The infected rhizomes resulted in 100% transmission and mechanical transmission failed to transmit the virus. Changes in total carbohydrates, chlorophyll, phenol, total soluble proteins and defense related enzymes namely peroxidase, polyphenol oxidase and phenylalanine ammonialyase were carried out at 30, 60, 90 and 120 days after infection. The study revealed an increase in the content of phenols and defense related enzymes in infected plants. An increase in carbohydrates, chlorophyll and protein in healthy plants was also observed. Protein profile study using sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS- PAGE) indicated the presence of a novel protein with molecular weight of 20 kDa in the infected plant sample. The immunological detection techniques direct antigen coating- enzyme linked immunosorbant assay (DAC- ELISA) and dot immunobinding assay (DIBA) were carried out. Since the etiology of the virus was unknown, seven suspected viruses were tested in DAC- ELISA and antibodies specific to two viruses namely Banana bract mosaic virus (BBrMV), Cucumber mosaic virus (CMV) gave an absorbance value of 0.15 and 0.19 respectively which was three times more than the absorbance shown by the healthy leaf (0.05 and 0.07). The infected leaf tested for the presence of African cassava mosaic virus (ACMV) by triple antibody sandwich ELISA (TAS-ELISA) gave an absorbance of 0.0425 while the healthy gave an absorbance of 0.017. DIBA analysis gave positive reaction to BBrMV. Polymerase chain reaction ( PCR) was carried out with both total DNA and RNA isolated from infected ginger leaf sample. The PCR experiment was positive to Begomoviruses and negative to PVY (Potato virus Y) and BBrMV isolates. An amplicon of size 550 bp was obtained for the sample DNA using begomo degenerate primer. The sequence was subjected to BLAST analysis which indicated 74 per cent similarity to Tomato leaf curl virus Bangalore isolate. The management studies were conducted with antiviral principles like botanicals, chemicals and that of microbial origin against the virus. The experiment was conducted in completely randomized design (CRD) with 13 treatments and three replications. Karthika was the variety used for the study. Perfekt (a botanical extract-76%) at 0.5 ml L-1 and 1ml L-1, chemicals namely aspirin, salicylic acid, barium chloride, at 100 and 150 ppm concentrations and botanicals namely ten per cent leaf extracts of Mirabilis jalapa and Bougainvillea spectabilis, two per cent neem oil garlic emulsion and two per cent PGPR mix II were used in the experiment. The treatments were given at fortnightly interval. Before each spray the efficacy of the treatments were evaluated using Vulnerability Index (V.I) developed by Bos (1982). The treatments with Perfekt at the rate of 0.5 ml L-1 and 1.0 ml L-1 and ten per cent leaf extract of Mirabilis jalapa were found effective for the management of the disease. The study indicates that the mosaic disease in ginger is transmitted through rhizomes, the virus has been molecularly characterized and shows only 74% identity with Tomato leaf curl virus (TLCV) and management of the disease can be done by application of Perfekt at 0.5 ml L-1 or 10% leaf extract of Mirabilis jalapa at fortnightly interval.ThesisItem Open Access Characterization, host range and management of papaya ringspot virus (PRSV)(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2018) Atheena Harish; KAU; Anita Cherian, KPapaya is an important fruit crop which is cultivated extensively both in tropics and subtropics. During the last decade, the area under papaya cultivation has dramatically increased due to the introduction of superior varieties. A major setback subsequent to the introduction of such new varieties has been the incidence of papaya ringspot disease caused by Papaya ringspot virus (PRSV) which leads to almost 100 per cent yield loss. Considering the importance of the disease, the project was undertaken with the objectives of characterization of the virus, molecular and immunodiagnosis along with disease management. The research project was initiated with purposive sampling surveys conducted in different papaya orchards of Thrissur district in order to document the symptoms under natural conditions, to assess the incidence and severity of the disease and to collect infected samples for further studies. The maximum disease incidence and disease severity recorded were 99.6 and 96.67 per cent on papaya variety Red Lady from Vellikulangara and Puthur respectively. The development of symptoms was studied under artificial conditions also through mechanical inoculation of healthy papaya seedlings with virus inoculum maintained in insect proof net house conditions. The salient diagnostic symptoms of the disease observed on leaves were chlorotic spots, mottling, vein thickening, puckering, leaf distortion, shoestring symptom along with presence of oily streaks on the petiole. The fruits of infected plants presented typical oily concentric or broken ringspots on fruit surface along with malformation. Histopathological studies of infected leaves revealed disruption of the epidermis, disorganization of parenchyma, disintegration of chloroplasts and deposition of crystalline bodies. The studies on virus transmission confirmed that it is transmitted through plant sap from infected to healthy papaya plants. Seed transmission studies revealed that PRSV is not seed -borne. Twenty one plant species including weeds seen in and around papaya orchards were tested for studying the host range of the virus and only five plant species viz., Cucumis sativus, Cucurbita moschata, Trichosanthes cucumerina, Momordica charantia and Chenopodium amaranticolor developed symptoms after artificial inoculation of PRSV and thus proved to be the hosts of the virus. Morphological characterization done using electron microscopy showed the presence of typical flexuous rod particles of size 807.74 nm x 12 nm which indicated that the virus belongs to genus Potyvirus and the etiology of the disease was confirmed as Papaya ringspot virus. Immunodiagnostic technique was validated using Direct Antigen Coating - Enzyme Linked Immuno Sorbent Assay (DAC-ELISA) and infected samples showed positive reaction to PRSV antiserum and could be detected at 1:200 dilution of primary antibody and 1:10,000 dilution of secondary antibody. Molecular characterization of PRSV was also carried out through Reverse Transcription Polymerase Chain Reaction (RT-PCR). The nuclear inclusion b (NIb) gene and the coat protein (CP) gene were amplified using reported primer pairs which yielded amplicons of approximate size of 1700 bp. The PCR products were outsourced for sequencing and in silico analysis of the sequences obtained revealed that the isolates of present study are more similar to Calicut isolate of PRSV, PRSV- Ca (DQ666640.1) A pot culture experiment under insect proof conditions was also conducted to evaluate the effects of selected botanicals, chemicals and biocontrol agents for disease management. Among the fourteen treatments, Bougainvillea leaf extract, 10 per cent (T7) was the most effective with lowest disease severity (6.67%) followed by foliar spray and soil drenching with Pseudomonas fluorescens 2 per cent (T3) with a disease severity of 11.11 per cent as against 97.77 per cent recorded in untreated control plants. Plant height recorded after each treatment application revealed that both 10 per cent Bougainvillea leaf extract (T7) and 2 per cent P. fluorescens are equally superior to all other treatments. However, with respect to mean girth of stem, maximum value (2.91 cm) was recorded in P. fluorescens (T3) The data on virus titre of all treated plants assessed through DAC-ELISA revealed that the concentration of virus particles was minimum in plants treated with T7 and T3. The outcome of this study would facilitate early detection and elimination of source of virus infection and thereby prevent the spread of disease in the field. The information generated on molecular characterization of PRSV isolates under could be applied in genetic engineering. The project also revealed the potential of botanical, Bougainvillea spectabilis leaf extract and biocontrol agent, Pseudomonas fluorescens for the ecofriendly management of papaya ringspot disease.ThesisItem Open Access Triazole,strobilurin and its combination fungicides for the management of anthracnose and fruit rot of chilli(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Anjana, R S; KAU; Joy, MA study entitled ‘Triazole, strobilurin and its combination fungicides for the management of anthracnose and fruit rot of chilli’ was conducted during 2016 - 18 at Department of Plant Pathology, College of Agriculture, Vellayani with the objectives to study the host range of the pathogen Colletotrichum capsici and to develop management strategy to control the disease using new generation fungicides. A survey was conducted at Vellayani, Kumarakom, Thrissur, Pattambi, Ambalavayal and Padanakkad during 2016 – 17 to screen the most virulent isolate of C. capsici in chilli (Capsicum annuum). During the survey period, C. capsici was found causing the disease in five and C. gloeosporioides in one locations. The maximum disease incidence (70 %) and severity (45 %) by C. capsici was recorded from Padanakkad. Except from Ambalavayal, five pure cultures of C. capsici (C1 to C5) were obtained and Koch’s postulate is proved. C. capsici produced white to dirty white, sparse mycelial growth on PDA, which later turned into dirty grey colour. Days taken to cover petridish (9 cm radius) ranged from 7.4 to 8.3 days. Mycelium was hyaline, septate and branched. Black colored acervuli were produced after 20 - 30 days of incubation and its diameter ranged from 21.85 - 45.82 μm, with 18 - 47 dark setae of 72.50 - 110.41 μm length. Conidiophores are hyaline, short and cylindrical with sickle shaped single celled conidia having an oil globule at the centre. The average size of the conidia was 20.04 - 23.48 μm x 2.58 - 3.22 μm. Based on screening of isolates through leaf and fruits inoculation of chilli variety ‘Vellayani Athulya’, C3 was identified as the most virulent isolate. The identity of C. capsici (C3) was confirmed by PCR using ITS primers, sequencing of amplicon, BLAST and phylogenetic analysis. The best solid and liquid media for growth and sporulation of C. capsici was potato dextrose medium, the optimum temperature was 30oC and pH was 6.5. The growth of C. capsici was studied under different light conditions and the fungus growth was maximum in 8 hour light and 16 hour dark under white light at 20 lux intensity. Host range of C. capsici was studied in different vegetable crops viz. Capsicum chinense, C. frutescens, cowpea, brinjal, tomato and bhindi. C. capsici infected all the host plants tested and brinjal was found as the ideal host plant. Among eight KAU varieties of chilli viz. Vellayani Athulya, Jwalamukhi, Jwalasakhi, Ujwala, Anugraha, Keerthi, Vellayani Thejus and Vellayani Samrudhi, screened against C. capsici with leaf and fruits inoculation, Vellayani Athulya was found to be the most susceptible variety to the disease, whereas Vellayani Thejus and Vellayani Samrudhi were found comparatively resistant to leaf and fruit infection. Under in vitro evaluation of new generation fungicides by poisoned food technique, azoxystrobin 11 % + tebuconazole 18.3 % SC at 10 ppm, pyraclostrobin 20 % WG at 50 ppm and hexaconazole 5 % EC + pyraclostrobin 20 % WG at 100 ppm completely inhibited the mycelial growth and proved to be most effective. All fungicides significantly inhibited spore germination of C. capsici. Hexaconazole 250 ppm completely inhibited germination. Pyraclostrobin 20% WG at 10 ppm, hexaconazole 5 % EC + pyraclostrobin 20 % WG at 50 ppm, tebuconazole 25.9 % EC and hexaconazole 5 % EC at 100 ppm were also equally effective in inhibiting germination of spores and were statistically on par. A pot culture study was conducted with chilli variety ‘Vellayani Athulya’ to evaluate the efficacy of selected new generation fungicides. The selected treatments were imposed only once, after 18 days of flowering when at least 25 per cent of fruits were at maturity stage. At recommended dose, the maximum disease suppression was recorded with 0.24 % of copper oxy chloride 50 % WG (90.59 %), followed by 0.1 % of hexaconazole 5 % EC + pyraclostrobin 20 % WG (73.50 %), 0.15 % of azoxystrobin 11 % + tebuconazole 18.3 % SC (70.94 %) and 0.1 % of tebuconazole 25.9 % EC (68.70 %). At higher (double the recommended) doses of respective fungicides, disease suppression was more. Biocontrol agents were also effective, but the disease suppression was less with 22.22 per cent for Trichoderma viride and 43.58 per cent for Pseudomonas fluorescens compared to the new generation fungicides. Hence anthracnose and fruit rot of chilli can be effectively managed by foliar spray of hexaconazole + pyraclostrobin combination fungicide and it may be subjected to mutlilocation and multiseasonal trials before recommendation.ThesisItem Open Access Characterization and management of yellow mosaic disease of black gram (vigna mungo (L.) hepper)(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2018) Divya Jayakumar, V J; KAU; Sumiya, K VBlack gram (Vigna mungo (L.) Hepper) is one of the important pulse crop in India and an excellent source of good quality protein. Yellow mosaic disease (YMD), caused by a whitefly transmitted geminivirus is the major constraint for black gram cultivation. Yellow mosaic disease of pulses is extensively studied from different parts of the country. But no study has been conducted in Kerala. Hence, the present study was undertaken to characterize the virus causing yellow mosaic disease in black gram in Kerala and to evolve suitable strategies for its management. Purposive sampling surveys were conducted in black gram growing areas of Palakkad and Malappuram districts, covering 13 fields at nine locations to study the incidence and symptomatology of yellow mosaic disease. The disease incidence in the fields varied from 12 to 100 per cent. Common symptoms observed in the field were typical yellow mosaic, puckering and cupping of leaves, distortion of leaf lamina and drastic reduction in size of younger leaves. Irregular whitish discolouration of leaves which turned papery white on maturity was also observed in some fields. Complete yellowing of leaves along with brown discoloration between the veins and vein banding symptoms which were not reported by earlier workers were also observed in the field. Electron microscopic studies revealed the presence of geminate particles of 15-18 x 30 nm size in infected black gram samples suggesting the association of a geminivirus with the disease. Whitefly transmission of the virus to healthy black gram plants was attempted and 80 per cent transmission was achieved. PCR amplification using virus specific primers revealed the presence of Mungbean yellow mosaic virus (MYMV) and Horsegram yellow mosaic virus (HYMV) in the samples. MYMV was detected in infected samples from nine fields out of the 13 fields surveyed. HYMV was detected in six fields, which include five fields in which MYMV was also detected revealing the occurrence of mixed infection in the field. Five representative isolates were sequenced at Agrigenome Labs, Kakkanad, Ernakulam. In silico analysis of these sequences revealed that the coat protein region of the isolates showed more than 90 per cent homology with MYMV isolates. This confirmed the presence of MYMV as the major virus in yellow mosaic disease of black gram in Kerala. Phylogenetic analysis revealed that the isolates from the present study are more closely related to MYMV isolates from southern parts of India and distantly related to Mungbean yellow mosaic India virus (MYMIV) isolates, which were reported from northern parts of India. Host range studies conducted in insect proof cages under glass house condition showed that the virus could be transmitted through whiteflies only to horse gram. Symptoms were observed in horse gram and Synedrella nodiflora, a predominant weed found in the field 20 -25 days after inoculation. But the presence of virus was confirmed by PCR only in symptomatic horse gram and not in Synedrella nodiflora. A field experiment was conducted during Rabi 2017-18 at RARS, Pattambi and evaluation of effectiveness of botanicals, biocontrol agents and other organic products revealed that application of Pseudomonas fluorescens as seed treatment @ 10g/kg seed and foliar sprays @ 10g/l at fortnightly intervals starting from 15 days after sowing or foliar sprays of 10 per cent aqueous extract of leaves of Bougainvillea spectabilis or roots of Boerhaavia diffusa at fortnightly intervals starting from 15 days after sowing are effective in reducing the yellow mosaic disease. The present study reveals that MYMV is the virus associated with the YMD of black gram in Kerala and it can be effectively managed by prophylactic application of Pseudomonas fluorescens, 10 per cent leaf extract of Bougainvillea spectabilis or 10% root extract of Boerhaavia diffusa. This is the first report on identification of MYMV associated with yellow mosaic disease of black gram in Kerala.ThesisItem Open Access Characterization and management of powdery mildew of yard long bean (vigna unguiculata subsp.sesquipedalis (L.) verdc.) under protected cultivation(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2018) Rahila Beevi, M H; KAU; Sainamole Kurian, PYard long bean (Vigna unguiculata subsp. sesquipedalis (L.) Verdc.) is believed to be selected and developed from cowpea (Vigna unguiculata (L.) Walp.) for its long, succulent pods which are used as a vegetable. In Kerala, it is one of the most preferred vegetables having very high amount of protein, iron, calcium, vitamin A, Vitamin C and dietary fibre. It is considered as a remunerative crop under protected condition owing to its high market demand. However, incidence of diseases is a major setback hampering the production of yard long bean under protected conditions among which, powdery mildew is the most devastating one. In this background, the present study was undertaken to characterize the pathogen causing powdery mildew of yard long bean and to formulate a management strategy for the disease under protected cultivation. Purposive sampling surveys were conducted in seven locations of Thrissur district and the disease severity varied from 1.67 to 67.33 per cent. The results of the survey indicated that the severity of disease was more during pod bearing and harvesting stage. Since powdery mildews are obligate parasites, characterization was done based on the microscopic observation of pathogen present on the leaves. The fungi produced hyaline, branched and septate hyphae. The conidiophores were erect and cylindrical on which conidia were born in chains. Variability was observed regarding conidia and conidiophore characters of powdery mildew collected from different locations, based on which the isolates were grouped into two viz., PM 1 and PM 2. PM1 type was observed in all locations except Vellanikkara. Based on the morphological characteristics of conidia and conidiophores, it was identified as Erysiphe polygoni. PM 2 type obtained only from Vellanikkara in which conidia and conidiophore characters were similar to Podosphera sp. which is very rarely reported on legumes. Hence, its identity was further confirmed as Podosphaera xanthii by molecular characterization. The rRNA-ITS sequence was deposited in NCBI Genbank database with accession number MH645799. This is the first report of powdery mildew of yard log bean incited by Podosphaera xanthii. In-vitro evaluation of 17 treatments including fungicides, biocontrol agents and botanicals by spore germination technique revealed that all the treatments caused cent per cent inhibition of conidial germination. For taking forward eight promising treatments to the field experiments, they were tested in-vitro on detached leaves by artificial inoculation of conidia from infected leaves. Based on the per cent leaf area infected, two systemic fungicides, one contact fungicide, two biocontrol agents and one botanical were selected for field evaluation. Field experiments were conducted simultaneously inside polyhouse and rain shelter to evaluate the performance of selected fungicides, biocontrol agents and botanicals against powdery mildew. Among the treatments, low disease severity of 4.33 per cent and 7.67 per cent was recorded in T1- difenoconazole and T2 – tebuconazole respectively in polyhouse and these treatments were statistically on par. In rain shelter also, T1- difenoconazole and T2- tebuconazole recorded low disease severity of 7.67 per cent and 10.67 per cent respectively. The performance of wettable sulphur at lower and higher concentration did not differed significantly. All the four non-chemical treatments were equally effective in managing the disease both in polyhouse and rain shelter. Correlation analysis between the meteorological parameters and disease severity revealed that per cent disease severity was negatively correlated with temperature and relative humidity both in polyhouse and rain shelter. Analysis of population of phylloplane microflora showed that, there was a drastic reduction in the population of phylloplane fungi and bacteria after spraying chemical fungicides which is an indication of the toxicity and non-selectivity of these chemicals. Survival ability of biocontrol agents sprayed on the leaves were studied and found out that both Trichodema viride and Pseudomonas fluorescens survived on the leaves for seven days. Residue analysis of difenoconazole, the most effective chemical fungicide revealed that the compound with initial deposition of 0.21 mg kg-1 dissipated to 0.09 mg kg-1 after seven days in polyhouse whereas, the residue after seven days in rain shelter was 0.19 mg kg-1. The faster degradation of the chemical inside polyhouse may be attributed to the higher temperature prevailed during the experiment. Evaluating the results various experiments in the present investigation, it was found that, even though chemical fungicides provided best disease control, considering their toxic effect on beneficial non target microflora on the phylloplane and the residue left on edible pods, biocontrl agents such as Trichoderma viride and Pseudomonas fluorescens which exhibited consistent performance with moderate disease control and sufficient survival on the leaf surface would be ideal to control powdery mildew of yard long bean if applied at right time. Moreover, frequent application of systemic fungicides with single site action can result in the development of resistant strains of pathogens. So such chemicals should be adopted only if the disease severity is very high and cannot be managed with biocontrol agents.ThesisItem Open Access Exploitation of abiotic stress tolerant strains of trichoderma spp. for the management of soil borne fungal pathogens(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2018) Stella Doncy, P P; KAU; Reshmy VijayaraghavanSoil borne phytopathogenic fungi are known to cause severe yield loss of several crops. To mitigate these crop ailments, farmers mostly rely on chemical methods of disease control such as fungicides and other pesticides which are deleterious to the environment. To date, biological control is an ecofriendly approach for the effective management of crop diseases. The fungi belonging to the genus Trichoderma are one among the most exploited biocontrol agents in the field of agriculture. However, the performance of Trichoderma spp. gets reduced when it is exposed to abiotic stressed conditions such as drought, high temperature, salinity, acidity and fungicides. Hence, this study was proposed to identify and exploit stress tolerant isolates of Trichoderma spp. with antagonistic potential in Kerala. Intensive soil sampling surveys were conducted across different stressed ecosystems of Kerala viz., Palakkad, Alappuzha, Vytilla, Kumarakom, Wayanad and Thrissur for the isolation and enumeration of native Trichoderma spp. A total of 24 isolates were obtained from 52 soil samples collected from different locations. Based on the number of Trichoderma spp. obtained from each district, they were serially numbered and abbreviated according to the name of the location. Accordingly, PAT 1 to PAT 6 represents number of isolates of Trichoderma spp. from Palakkad district, ALT 1 to ALT 3 from Alappuzha, VYT 1 and VYT 2 from Ernakulam district, KUT 1 from Kottayam district, WAT 1 to WAT 8 from Wayanad and THT 1 to THT 4 from Thrissur district. Cultural and morphological identification of these isolates were carried out under in vitro conditions. Isolates of Trichoderma spp. were subjected to in vitro screening for abiotic stress tolerance such as high temperature, drought, acidity, salinity and also to test their sensitivity towards copper fungicides. The isolates PAT6 and WAT2 were found as thermotolerant, VYT2 and ALT 1 as drought tolerant, ALT 3 and ALT 1 as acid tolerant and saline tolerant and the isolates ALT1, ALT3 and PAT 1 as copper fungicide tolerant. The selected six isolates were further subjected to biochemical tests and the study showed that the isolates VYT 2, ALT 3 and ALT 1 showed highest cellulase, β- 1, 3 glucanase and protease activity. Likewise, isolates PAT 1, ALT 1 and ALT 3 were found as best producers of ACC deaminase and PAT 1 and ALT 1 as the best cytokinin producer. The best performing isolates (ALT 1, ALT 3, PAT 1, PAT 6 and VYT 2) after enzyme study were subjected to dual culture experiment with five major soil borne pathogens to test their antagonistic potential. The isolates ALT 3, ALT 1, PAT 6 and PAT 1 showed more than 70 per cent inhibition of R. solani whereas, isolates ALT1 and VYT 2 showed only 57.78 and 55 per cent inhibition of S. rolfsii respectively. However, no significant difference was noticed among the isolates when grown against F. solani. Cent per cent inhibition of P. aphanidermatum was noticed with Trichoderma isolates PAT 1 and ALT 1. All five isolates showed 100 per cent inhibition on the growth of pathogen, P. capsici. Among the five, four isolates viz., ALT 1, ALT 3, PAT 1 and PAT 6 with best antagonistic potential were subjected to molecular characterization at Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram and the isolates showed homology with the nucleotide sequence of Trichoderma asperellum. A pot culture experiment was laid with the selected isolates viz., ALT 1, ALT 3, PAT 1 and PAT 6 to test the growth promotion of cowpea and biocontrol efficacy against Rhizoctonia solani. It was observed that the isolate PAT 6 coated seeds showed 100 per cent germination and also recorded better biometric characters and yield. Moreover, the lowest per cent disease incidence of 11.11 per cent was only recorded with both the isolates ALT 3 and PAT 6. Thus, the study has enlightened our knowledge on the existence of abiotic stress tolerant isolates of T. asperellum which can be employed in future for the biocontrol of soil borne pathogens in such conditions.ThesisItem Open Access Ecofriendly management of anthracnose of betel vine (piper betle L.)(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Nisha, A; KAU; Heera, GThe study entitled "Ecofriendly management of anthracnose of betel vine" was conducted during 2016-18 at Department of Plant Pathology, College of Agriculture, Vellayani with an objective to evolve an integrated management strategy involving organic preparations, bio control agents and new generation fungicides for anthrancnose of betele vine. A survey was conducted in different locations in Southern Kerala viz., Thiruvananthapuram (Kalliyoor, Vellayani and Kattakada), Kollam (Kareepra), and Alappuzha (Cherthala) regions during 2016-18. Anthracnose was the major problem in these areas. The highest percentage disease index of 20.00 was observed from Cherthala region of Alappuzha. The symptoms of the anthracnose appear as small necrotic spots with a yellow halo on the leaf lamina mostly starting from the margin of the leaf. These lesions coalesce together results in leaf blighting and defoliation. Under high humid conditions, stem lesions were also produced which resulted in drying of the entire vines. Colletotrichum sp. was found to be associated with the disease in five locations. Isolations were made and five pure cultures of Colletotrichuym sp. (C1 to C5) were obtained and its pathogenicity was proved using Koch's postulates. Culture and morphological characters of the five different isolates were studied. The isolated cultures of Colletotrichum sp. produced whitish to dark grey, orangish to off white coloured colony having fluffy to sparse mycelial growth with regular margins. The different isolates took 7-9 days for completion of growth in petri dish. The mycelium of the fungus was hyaline,septate and width ranged from 2.65 - 3.45µm. The septal distance varied from 9.80 - 25.56 µm. The conidia were single celled with an oil globule and the shape varied from cylindrical, oblong to dumbbell among the isolates. The condial and appressorial size varied from 9.6-12.2 µm *3.8 - 4.7 µm and 8.37 - 10.08µm *5.02-6.28µm. The pathogenic variability among the five different isolates were assessed by virulence rating. The isolate C2 was identified as most virulent which produced lesion size of 14.75 mm at 5 days after inoculation (DAI). The isolate C2 initiated symptom within two days of artificial inoculation having a rate of lesion development of 2.95 mm day-1. The identity of C2 isolate as C. gloeosporioides was confirmed by the use molecular techniques (DNA barcoding). The indigenous organic preparations viz., panchagavya, jeevamruth, vermiwash and fermented cow’s urine at 5 and 10 per cent (autoclaved and non-autoclaved) were evaluated for in vitro pathogen suppression. The micronbial population was estimated at 1, 5 and 7 days in the filtered and non-filtered indigenous organic preparations. Microbial population of bacteria was comparatively high in these organic preparations when compared to fungi. The bacterial population showed an increasing trend in non-filtered organic preparations except jeevamruth. The mycelial nature in amended media varied from fluffy to sparse growth with uniform or irregular growth pattern under autoclaved and non-autoclaved extracts of organic preparations. Panchagavya at 10 per cent and fermented cow's urine at 5 percent and 10 perecent (non-autoclaved) completly inhibited the pathogen. Autoclaved fermented cow’s urine at 5 pere cent and 10 per cent failed to inhibit the mycelial growth of the pathogen. The autoclaved and non-autoclaved organic preparation panchagavya at 10 per cent was more effective than other treatments in inhibiting the mycelial growth of the pathogen. The saprophytic micro flora were isolated form the rhizosphere and phyllosphere of healthy betel vine. The fungal and bacterial biocontrol agents from phyllosphere and rhizosphere were screened in vitro for pathogen suppression. The fungal antagonist Trichoderma 1 was superior in inhibiting the mycelial growth of C. gloeosporioides. Among the bacterial isolates screened, the isolate B5 from the phyllosphere of the healthy betel leaves had a higher percentage of mycelial inhibition (45.00 per cent). Screening of fungicides against the fungal pathogen under in vitro condition revealed that systemic fungicides (Propiconazole, tebuconazole), combination fungicides (captan + hexaconazole and carbendazim + mancozeb) at 0.05, 0.1 and 0.2 per cent completely inhibited the mycelial growth of the pathogen. The contact fungicide copper hydroxide was also effective inhibiting the mycelial growth. The fungicide Propiconazole was effective in suppressing complete growth of the pathogen at lower dose of 0.01 per cent (100 ppm). A pot culture study was laid out in CRD with 8 treatments in 4 replications to develop an integrated management package. The effective treatments of the above studies viz., panchagavya (10 per cent), bacterial isolate (B5-108 cfu/ml), propiconazole (0.1 per cent), copper hydroxide (0.2 per cent)carbendazim and mancozeb (0.2 per cent) and chemical control check (Bordeaux mixture - 0.5 per cent) with inoculated and un inoculated control were evaluated for the management of anthracnose of betel vine. The treatment of propiconazole was superior in reducing the percentage disease index and disease incidence. The treatment recorded a disease index of 19.99 per cent at 10 days after second spry with 74.99 per cent disease suppression over rinoculated control. The second best treatment was the foliar application of carbendazim + mancozeb (0.2 per cent) which was on par with copper hydroxide (0.2 per cent) in reducing the severity of the disease. Even though the chemicals were far more effective in managing the disease, the bacterial isolate B5 was equally effective in suppressing the pathogen with 46.24 per vent disease suppression over inocu;lated control with d disease index of 42.98. The biometric observations did not show significant difference among the different treatments except the number of leaves. The present study reveals that all the fungicides and bacterial isolate from phyllosphere were effective in managing the disease over the traditional management using Bordeaux mixture at 0.05 per cent POP of KAU, 2016 since it is a masticatory crop need based application of antagonistic bacteria may be suggested for the anthracnose management in betel vine. For proper disease management approach, the bacterial antagonist may be suggested after conducting further studies on the spray schedule, dosage compatibility with fungicides and its biosafety.ThesisItem Open Access Characterization of medicinal mushroom, cordyceps sp. from Kasargod district(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2018) Laya, P K; KAU; Yamini Varma, C KCordyceps which is an entomo-pathogenic fungus got the name from two Latin words, cord and ceps, meaning ‘club’and ‘head’. This mysterious fungus has a long reputation of being the single-most expensive raw material used in traditional Chinese medicine due to its dearth in availability. As a result of its high efficacy and potency in curing various diseases, this fungus has been recommended by medicinal practioners as a ‘panacea of all ills’. Distribution of Cordyceps sp. is cosmopolitan, but mostly confined to high Himalayan Mountains in China, Tibet, Nepal and India, at an altitude of 3000 to 5000 metres or in Asian high altitude grass land ecosystems. Cordyceps sp. which is renamed as Ophiocordyceps sp. attacks on the coconut root grub (Leucopholis coneophora Burm.) in coastal sandy areas of Kasargod district of Kerala. In this context, in the present study, ‘Characterization of medicinal mushroom, Cordyceps sp. from Kasargod district’ an attempt was made to untangle many of the mysteries of Cordyceps sp. by isolation, identification, characterization, cultivation and assessment of the proximate composition with special attention to Cordyceps sp. Purposive sampling surveys were conducted in three locations of coastal sandy tracts of Kasargod district during June to September months of 2017 and 2018. The fruiting bodies of Cordyceps sp. were collected from three locations viz., Instructional farm of College of Agriculture, Padannakkad, Valiyaparamba area of Nileshwar and Regional Agricultural Research Station, Pilicode. Maximum number of fruiting bodies were obtained from the soil under cashew trees in the Instructional farm of College of Agriculture, Padannakkad (Average of 13.86/m2) due to the richness in organic carbon (1.39%), and shaded condition in the soil under cashew orchard compared to Valiaparamba and Pilicode. Macro minerals viz., potassium, calcium and micro mineral like, manganese were in higher quantity under soil of cashew trees compared to other two regions, which may be helping in the survival and germination of spores. The fungus was isolated on PDA medium from the stroma portion of the fruiting body and the isolate obtained after pure culturing was numbered as CD-1. The isolate completed the full growth in a 9 cm Petri dish by 55 days after inoculation in PDA medium. The anamorph produced similar type of conidia as produced in the field. Characterization of Cordyceps sp. based on cultural, morphological and physiological characters were carried out. The cultural characters like colour, shape, texture and the growth rate of the mycelium were studied in different media. Among the different media tested yeast potato dextrose agar medium (YPDA) was found to be the best medium followed by potato dextrose agar media (PDA), which were significantly superior from other medium. Various macroscopic characters like shape, size and colour of sclerotia, stipe and stroma and branching pattern of the stroma in three locations were observed. Microscopic studies of both anamorph (hyphae, synnemata, conidiophores and conidia) and teleomorph (perithetia, asci and ascospores) were conducted. The molecular characterization of the fungus was carried out at Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananthapuram and showed homology with Ophiocordyceps neovolkiana (Kobayasi) strain KC1 having 98 per cent identity. The sequence of the fungus was deposited at the Gen Bank of NCBI and the accession number obtained was MH 668282. The culture was deposited in National Fungal Culture Collection of India at MACS Agharkar Institute, Pune with culture no. NFCCI 4331. Physiological characterization of O. neovolkiana was carried out. Among eight different temperatures tested, 30oC was found to be the optimum temperature in which the growth was completed within 38 days after inoculation. The neutral pH seven was found to be optimum for mycelial growth. Among the different light conditions, 24 hrs of darkness under incubator (30oC) was preferred by this fungus. Fructose was the best source of carbon in which maximum mycelial growth was obtained within 36 days after inoculation. The best source of nitrogen was yeast extract in which maximum mycelial growth was obtained within 35 days after inoculation. KH2PO4 was the best source of macro mineral for the growth, in which the fungus completed the growth within 38 days after inoculation. Among micro minerals, ZnCl2 was the best for the growth in which the fungus completed the growth within 35 days after inoculation. Folic acid was the best source of vitamin for the growth in which the fungus completed the growth within 37 days after inoculation. One optimum media for the growth of O. neovolkiana was standardized as Yeast Potato Fructose medium supplemented with one gram of KH2PO4, 500 mg of ZnCl2, 10 mg of folic acid in one litre of distilled water which was named as Yeast Extract Potato Fructose Agar (YEPFA). The growth of O. neovolkiana in the YEPFA medium was significantly higher than the best media YPDA and PDA, so that the time required for the mycelial growth could be considerably reduced from 55.15 days to 30.85 days. The mycelial dry weight was also significantly higher in YEPFB medium than YPDB and PDB and growth completed within 32.57 days after inoculation. Different cereal grains viz., rice, wheat and sorghum were tried for artificial culturing of O. neovolkiana under in vitro conditions. The number of days taken for coverage of the mycelium in 50g of grains was 96.4 days for sorghum which was significantly different from rice (115.2 days) and wheat (142.2 days). Hence sorghum was selected as the best substrate for artificial culturing. Analysis of proximate constituents was estimated using the collected sporocarps from the field and mycelial growth obtained on the broth of YEPF medium. The carbohydrate was more in artificially cultivated mycelia than fruiting bodies (5 and 9.7% respectively). But protein, (2.9 and 1.75%) and vitamin C (3.27 and 2.5) were more in fruiting bodies than mycelia. The crude fibre was more in mycelia than the fruiting bodies (16.8 and 25%). The total minerals content was almost similar in fruiting bodies and mycelia. Present work is the first attempt in India, which resulted in a detailed systematic study on O. neovolkiana parasitizing on coconut root grub. Future line of work should be concentrated on mass production of this fungus, the estimation of its anti- cancerous components and the antifungal and antibacterial effect of this fungus on pathogens in plant disease management and its potential as a biocontrol agent in pest management.ThesisItem Open Access Evaluation of biochemical and anti-cancerous activities of mushrooms(Department of Plant Pathologoy, College of Agriculture vellayani, 2018) Agnes Jose; KAU; Geetha, DThe present study on "evaluation of biochemical and anti-cancerous activities of mushrooms" was conducted during 2016-2018 with the objective to undertake the cultural, spawn production and cultivation trials of five mushrooms namely, Ganoderma lucidum (Curtis) P. Karst., Pleurotus florida (Mont.), Pleurotus djamor (Fr.) Boedjn, Hypsizygus ulmarius (Bull.:Fr.) Redhead and Calocybe gambosa (Fr.) Singh and to evaluate their biochemical and anti-cancerous activities. The cultures of these mushrooms were isolated from the mushroom beds maintained in the mushroom unit of Instructional Farm, Vellayani through tissue culture method and purified by hyphal tip method. Studies on mycelial growth of five mushrooms on potato dextrose agar revealed that G. lucidum had the maximum radial growth (9.00 cm) in shorter period of time (6.25 days). The nature of mycelial growth of G. lucidum and P. florida was flat and filamentous, while that of P. djamor, H. ulmarius and C. gambosa was thick and fluffy. The colour of mycelium of all the mushrooms was white to creamy white. Spawn production trials of five mushrooms on paddy grain indicated the minimum time for spawn run of 12.25 days for P. djamor followed by H. ulmarius and P. florida. Cultivation trials of P.djamor, P. florida, H. ulmarius and C. gambosa were undertaken on paddy straw substrate while that of G. lucidum was done on rubber wood sawdust. P. djamor recorded minimum time for spawn run (10.50 days), pinhead formation (14.50 days) and first harvest (16.50 days). The milky mushroom, C. gambosa recorded the maximum yield of 1037.25 g kg-1 from three harvests with 103.72 per cent biological efficiency (BE) followed by H. ulmarius with yield of 960 g kg-1 and BE of 96 %. Infestation of pests viz. phorid flies and staphylinid beetles as well as fungal contaminants such as Trichoderma sp., Aspergillus sp., and Coprinus spp. were found in all the mushrooms during sporocarp formation. Dried and powdered samples of mushrooms were used for nutritional analyses. G. lucidum recorded maximum protein (30.91 %) and fibre content (49.33 %), whereas C. gambosa recorded maximum carbohydrate, total amino acids, ash and fat content. The present study indicated the high nutritive value of milky mushroom C. gambosa Mineral components like sodium, potassium, phosphorus, calcium and magnesium were determined in the study. Among the five mushrooms C. gambosa recorded the maximum potassium while calcium and magnesium were the maximum in G. lucidum. The phosphorus content was not found to be significantly different among the five mushrooms. Analysis of medicinal components of mushrooms indicated that mushrooms were rich in beta-glucan, polyphenols, flavonoids and terpenoid. G. lucidum, the king of medicinal mushrooms recorded maximum beta-glucan (38.58 %) and polyphenols (23.80 mg GAE g-1) followed by C. gambosa. Flavonoid and terpenoid contents were recorded maximum in C. gambosa followed by G. lucidum. The pink oyster mushroom, P. djamor recorded the maximum beta- carotene content (355μg g-1). Preliminary trials on the anti-cancer activities of mushroom extracts were conducted by direct microscopic studies and MTT assay. The results revealed that percentage viability of cervical cancer cell lines decreased with increase in concentration of mushroom extracts. However G. lucidum extract exhibited maximum cytotoxic effect on cancer cell lines even at lower concentration (200 μg ml-1) followed by C. gambosa. The present study indicated that all the five mushrooms are rich source of protein, carbohydrate, fibre and minerals. P. florida, P. djamor, H. ulmarius and C. gambosa were nutritionally and medicinally superior. The commercial cultivation as well as clinical studies of these medicinally important mushrooms must be undertaken in large scale.