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ThesisItem Open Access Management of bitter gourd mosaic by enhancing host resistance(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2015) Ashwini, K N; KAU; Vimi, LouisBitter gourd (Momordica charantia L.) is one of the important vegetable crops that occupy a pivotal position among fruit vegetables, particularly in south India. The fruits of this crop which have high commercial value and are being used for culinary preparations and various medicinal preparations. In spite of the economic importance of this vegetable, the research work carried out on protection of crop from viral disease is quite scanty. In many case, cent per cent mosaic incidence was recorded in the crop resulting in substantial economic loss. So the present study was focused on screening of bitter gourd accessions and management of bitter gourd mosaic by enhancing host resistance using defense inducers. The three different viruses causing mosaic in bitter gourd are cucumber mosaic virus (CMV), potyvirus and bitter gourd distortion mosaic virus (BDMV). As these viruses causes mixed infection in field, the separation of individual viruses was carried out using systemic indicator host plants. For separation of CMV and potyvirus, systemic indicator host plants used were cosmos and papaya respectively. BDMV was separated by white fly transmission. The pure cultures of viruses were maintained on the susceptible bitter gourd variety Preethi. The symptoms developed by different viruses were recorded under natural and artificial conditions were recorded CMV produced mosaic specks, yellow-green mosaic patches, leathery leaves and downward rolling of leaf margin. Symptoms of potyvirus infection were vein clearing, puckering, malformed leaf with reduced leaf size and rugosity. BDMV infection produced mosaic, puckering, leaf distortion, hairy growth on leaves and vines with reduction in leaf size and internodal length. For the screening of bitter gourd accessions against CMV and potyvirus, potassium phosphate buffer pH 7.0 was found to be the most suitable buffer. Among 22 accessions screened, three accessions viz., TCR 285, TCR 39 and TCR 53 were highly resistant to CMV; one accession Biliagala was highly resistant to potyvirus and 11 accessions viz.,TCR 285, TCR 39, TCR 493 ,TCR 416, TCR 492, TCR 494,TCR 380, TCR 202 and TCR 149, Green long and Biliagala were highly resistant to BDMV. The field experiment was undertaken with the objective of management of bitter gourd mosaic by using defense inducers. The three different defense inducers viz., salicylic acid 25 ppm, barium chloride 0.1% and Pseudomonas fluorescens 2 % were evaluated on the moderately resistant cultivar white long and susceptible variety Preethi. The mosaic symptom was recorded after 51 days of sowing in salicylic acid treated plants and after 40 days of sowing in control. A time gap of 5-10 days after spray of defense inducer was required for development of resistance in plants. The lowest disease severity was observed in cultivar White long treated with salicylic acid. The highest yield was recorded in Preethi treated with Pseudomonas fluorescens.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 Characterization and integrated management of Fusarium oxysporum f.sp. cubense (E.F. Smith) synder and hansen causing fusarium wilt disease of banana(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2020) Lishma, N P; KAU; Anita Cherian, KFusarium wilt of banana caused by the soil borne fungus Fusarium oxysporum f. sp. cubense (Foc) is a serious constraint to banana cultivation in Kerala. The fungal species constitute four pathogenic races, of which Race 1 is the prevalent one in our country and Race 4 is one of the emerging threats, though not reported from Kerala yet. The present study was undertaken to characterize the associated pathogenic races and to develop an integrated package for the disease management. The project initiated with purposive sampling surveys in various districts viz., Thiruvananthapuram, Ernakulam, Thrissur, Palakkad, Kozhikode and Wayanad representing different agroclimatic zones of Kerala. The per cent disease incidence (PDI) and the per cent disease severity (PDS) ranged from 1.52 to 43.65 per cent and 20.34 to 49.57 per cent. The correlation analysis of PDI with weather parameters showed a positive correlation with rainfall. However, it was negatively correlated with temperature. The study on symptoms under natural as well as artificial conditions showed characteristic external and internal symptoms. The number of days taken for complete wilting under artificial inoculation was 29.67 in Rasthali (AAB), 47.99 in Njalipoovan (AB), 31 in Kadali (AA) and 37.67 in Chenkadali (AAA). Among the thirty isolates of the Foc collected, twenty three isolates were from Rasthali variety, four isolates from Kadali, two isolates from Njalipoovan and one from Chenkadali. Studies on identification of Foc races with the differential host assay revealed that the varieties such as Cavendish (assay host to Race 4), Nendran (assay host to Race 4), Heliconia sp. (assay to Race 3) and Monthan (assay to Race 2) did not produce any type of symptoms whereas, all the isolates produced symptoms on Rasthali (assay host to Race 1) variety. A non polymerase chain reaction (PCR) based quick molecular diagnostic technique with loop mediated isothermal amplification (LAMP) assay was developed for the detection of Races of the pathogen. All isolates showed positive reaction to the LAMP assay for Race 1 and negative for Race 4. A PCR was also standardised for the confirmation of the races. It is concluded that all the isolates collected from different agroclimatic zones belonged to the Race 1 category of the pathogen only. Cultural and morphological characterization of the isolates revealed white coloured aerial mycelium with pink pigmentation and cottony and fluffy mycelial mat. The mycelial growth rate in half strength potato dextrose agar (PDA) medium ranged from 0.83 to 2.40 cm/day and the length and breadth of macroconidia and microconidia measured about 15.01 - 20.20 μm x 2.14 - 5.07 μm and 4.49 - 7.42 μm x 1.35 - 3.13 μm respectively. The inter-septal length and breadth of hyphae ranged from 16.14 to 22.94 μm and 4.22 to 6.57 μm respectively and the size of chlamydospores varied from 5.68 to 9.58 μm in diameter. The PCR based molecular characterization of isolates using ITS (internal transcribed spacer) primers produced single bands of size approximately 580 bp. In silico analysis of the sequences showed 96 to 100 per cent homology to Foc. Based on cultural, morphological and molecular characters, the pathogen was identified as Fusarium oxysporum f. sp. cubense. The screening of accessions maintained in the germplasm of Banana Research Station (BRS), Kannara was done to assess their disease resistance to Foc Race 1 and were grouped into six categories. Fifteen immune varieties viz., Attunendran, Zanzibar, Big Ebanga, Nedunendran, Nendran, BRS II, Thiruvananthapuram, Pachanadan I, Cultivar Rose, Pisang Lilin, Pisang Jari Buaya, Yangambi Km5, Grand Naine, Chinese Cavendish and Nendran Hybrid and four highly susceptible varieties viz., Cheriya Poovan, Valiya Poovan, Kadali and Rasakadali were identified. The estimation of biochemical parameters for the assessment of host plant disease resistance against Foc Race 1 revealed that the activity of total phenols and defense related enzymes was more in resistant varieties compared to susceptible varieties and the activity of reducing and non reducing sugars was more in susceptible varieties. An in vitro experiment was conducted for the evaluation of chemical fungicides, biocontrol agents and botanicals for control of the pathogen. The effective treatments from in vitro evaluation were carried over to pot culture and field experiments for the disease management. Among the various treatments, an integrated package comprising of Pseudomonas fluorescens + arbuscular mycorrhizal fungi and Trichoderma enriched cow dung + tebuconazole (T6) was proved to be the best for yield and disease management. It is concluded that the present study has enlightened our knowledge on characterization, race identification and management of Fusarium wilt pathogen infecting banana.ThesisItem Open Access Management of early blight disease of tomato (Solanum lycopersicum L.) under protected cultivation(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2020) Sumbula, V; KAU; Sainamole Kurian, PTomato (Solanum lycopersicum L.) is one of the most remunerative and widely grown vegetables all over the world. With the coordinated efforts of central and state governments, protected cultivation of tomato is now gaining popularity in Kerala. Despite being a versatile crop adapted to various agroclimatic regions and seasons, cultivation of tomato is constrained by various fungal, bacterial and viral diseases. Among the fungal diseases, early blight caused by Alternaria solani is the most common, destructive and widespread in all the tomato growing tracts. Fungicides and bioagents are commonly used to manage plant pathogens. But little is known about their effects on the non-target microbial communities that inhabit inside and outside the plant. Hence, it has become necessary to consider the effect of different fungicidal and bioagent treatments on target and non-target microbial communities while formulating disease management strategies. So, the present investigation was carried out with the objectives to formulate suitable management strategies against early blight disease of tomato under protected cultivation and to assess their impact on culturable and non-culturable microflora associated with the plant. Isolation of the pathogen from infected tomato leaf samples revealed the association of the fungus, Alternaria sp. and its pathogenicity was established by inoculating on threemonth- old tomato seedlings. Symptoms observed on leaves, shoot and fruits were almost same under both natural and artificial conditions. Cultural and morphological characters of pathogen was studied on potato dextrose agar (PDA). Initially, pathogen produced greenish brown mycelium and later turned to grey colour. Hyphae are septate and the colony has aerial topography and irregular rough growth patterns with concentric zonation. Sporulation was observed after six days of incubation and conidiophores were straight or flexuous brown to olivaceous brown in colour. The conidia are solitary straight or muriform or oblong, pale or olivaceous brown, length 40-110 μm and 7-15 μm thick with 2-8 transverse and 0-3 longitudinal septa. The cultural and morphological characters of the pathogen completely fit into the description of Alternaria solani by Alexopoulos et al. (1996). Hence, it is confirmed that the symptom observed on tomato leaves are those of early blight disease caused by A. solani. In vitro evaluation of fungicides and bioagents showed complete inhibition of the pathogen with propineb (0.1%, 0.2% & 0.3%), hexaconazole (0.05%, 0.1% & 0.15%), iprodione + carbendazim (0.1%, 0.2% & 0.3%), difenoconazole (0.075%), Trichoderma viride (KAU), T. viride (PGPM mix), T. harzianum (PGPM mix) and plant growth promoting microbial consortium (PGPM mix of KAU). Among the bacterial antagonists, Bacillus subtilis (endophyte from cocoa) showed maximum growth inhibition of the pathogen. All the three bioagents recorded earliness in seed germination and enhanced seedling vigour compared to the fungicidal treatments and control. The results of field experiment under polyhouse and rain shelter conditions showed that all the treatments are superior to control in early blight disease management, of which, spraying of iprodione + carbendazim (0.2%) and propineb (0.2%) were the best among fungicides and PGPM mix application was the most efficient among bioagents. Moreover, the highest yield was recorded from iprodione + carbendazim treated plants. Biocontrol treated plants showed better performance in overall plant vigour of which PGPM mix application was the most effective. Residue analysis showed that degradation rate of fungicides was more under polyhouse condition. Analysis of population of phylloplane and endophytic microflora proved that there was drastic reduction in microbial population after spraying with chemical fungicides whereas population increased after bioagent application. The study on survival of bioagents on tomato phylloplane revealed that both Pseudomonas fluorescens and T. viride, survived on leaf surface up to 15 days after foliar application. Analysis of fungicidal residue on tomato fruits revealed that, the degradation of fungicides was faster in polyhouse compared to rain shelter. Metagenomic analysis of microbial diversity on tomato leaves revealed that spraying of chemical fungicides reduces microbial population and diversity while bioagent application enhances the same. However, microbial community structure was changed in both cases. This study also enlightened the new mode of action for fungicides and bioagents besides their direct effect that is shifting the microbial community structure so that it provides greater resistance against the pathogen. Interestingly, metagenomic results also showed association of Cladosporium, Corynespora, Pseudocercospora along with early blight pathogen Alternaria on tomato leaves that otherwise remain undetected. Another important observation was Clostridium in tomato leaf samples except in PGPM mix treatment, suggesting the possibility of plants as alternate host for major human and animal bacterial pathogens. Hence, considering the effects of treatments on per cent disease severity both under polyhouse and rain shelter condition, residue analysis, phylloplane and endophytic microbial enumeration study and metagenomics analysis of microbial diversity, the present study recommends spraying of propineb (0.2%) as the best treatment among the tested fungicides and spraying of PGPM mix among biocontrol agents for the management of early blight disease of tomato under protected cultivation. Further system-level analysis of the complex interaction that governs outcomes among community members in the context of the plant host is required, in order to identify microbial interaction and selection processes for beneficial communities at different concentrations of fungicides and pathogen pressures.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.
