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ThesisItem Open Access Characterization and management of ganoderma lucidum inciting basal stem rot of coconut(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2012) Yunus, C; KAU; Beena, SThe present study on “ Characterization and management of Ganoderma lucidum inciting basal stem rot of coconut ” was undertaken in the Department of Plant Pathology, College of Horticulture, Vellanikkara during 2010-2012 with an aim to isolate the pathogen associated with the disease and to study the cultural, morphological and pathogenic characters of different isolates of the pathogen, symptomatology of the disease, host range and effective management of the pathogen using bio-control agents, phytoextracts and selected fungicides. Purposive sampling surveys were conducted and the occurrence of basal stem rot disease of coconut was observed through out Kerala. The isolation of pathogen from basidiocarps yielded eight isolates of Ganoderma sp. which produced fruiting body in saw dust- rice bran substrate. The pathogenicity of these isolates was tested and observed yellowing, drying and drooping of leaves of coconut seedlings inoculated with all isolates except the isolate GT- from Trivandrum. Basidiocarp formation was noticed only in one seedling inoculated with the isolate GV from Vellayani and reisolation of pathogen was done from this basidiocarp. Symptomatology of the disease under natural and artificial conditions was studied. Under field condition the typical symptom of BSR disease viz., yellowing and drooping of leaves, stem bleeding and basidiocarp formaton were observed in all surveyed areas but all the typical symptoms of disease were not observed under artificial condition. The cultural characters of all the isolates of pathogen were studied on four media viz., Potato dextrose agar, Czapek’s (DOX) agar, Richard’s agar and Soil extract agar media. All isolates produced white mycelial growth on all media but variations in texture, mycelial type, and colour change of mycelium, exudates production and formation of aberrant fruiting body were observed. PDA was found to be the best medium for the growth of pathogen in which all isolates recorded highest growth rate. The pathogen preferred a temperature range of 30-350C and neutral to acid pH of 5-7 for the growth. Slight variation in growth rate was observed under light and darkness. Basidiocarps showed variations in the morphological characters and were stipitate in all isolates except GC from Chirakkacode and GVe from Vettikkal, semicircular to conical shaped, yellowish red to reddish brown with smooth to waved margin, creamy white to brown pore surface, 4.4 – 12.0 x 2.6- 17.0 cm size, 1-10 mm pore length, 139- 254 x 122 – 190 μm pore diameter and 2-10 mm flesh thickness. Basidiospores were brown, ovate to ellipsoidal, truncated apex, double walled with inter wall pillars separating two walls. The size of these basidiospores showed variation in the range of 4.8-13 x 4.5-7.0μm with a spore index of 1.15-1.7. It was trimitic, with generative hyphae hyaline, thin walled, branched, septate and clamped. Reddish brown pigmented skeletal hyphae and colourless binding hyphae were noticed. Based on these observations the eight isolates of the pathogen were identified as Ganoderma lucidum (Leys) Karst. Regarding the in vitro management of the pathogen, two isolates of T. virens and one isolate of T. viride were isolated from rhizophere soil and were proved equally effective with the reference culture, T. viride and T. harzianum in inhibiting the growth of pathogen. Mycoparasitism and production of non volatile metabolites were found to be the mechanisms exhibited by the selected Trichoderma spp. The bacterial antagonists obtained from rhizosphere soil and the reference culture P. fluorescens recorded less than 50 percent inhibition on the growth except in cases of few isolates of the pathogen. It was observed that the selected bacterial antagonists were not much effective in inhibiting the pathogen compared to fungal antagonists. Among the phytoextracts, Azadirachta indica at 20 per cent concentration was found the most effective and recorded more than 50 percent inhibition on the growth of pathogen over control. It was followed by Musa sp. at 10 per cent concentration. The in vitro evaluation of fungicides showed that flusilazole, hexaconazole and iprobenphos at 0.2 per cent concentration were the most effective and recorded cent per cent inhibition on the growth of all isolates of pathogen. The study on the host range of G. lucidium revealed that the seedlings of arecanut, breadfruit, acacia and jack fruit showed yellowing and drooping of leaves and finally wilting of all the seedlings were observedThesisItem Open Access Exploitation of spent mushroom substrate as mulch for the management of rhizome rot complex disease of ginger(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2012) Remya, J S; KAU; Sheela Paul, TSpent mushroom substrate (SMS) is the composted organic material remained after the harvest of a crop of mushroom and it is rich in plant nutrients including minerals and was used as manure in different crops including ginger. Recent findings illustrate that substrate after one cycle cultivation of mushroom can be used for disease management. So far no attempt has been made on the use of spent mushroom substrate from oyster mushroom, against rhizome rot complex disease of ginger. Under these circumstances, a study was conducted to know the effectiveness of oyster mushroom SMS to control the rhizome rot complex disease of ginger. Bacterial wilt and soft rot are the two common rhizome rot diseases of ginger. Ginger plants showing typical wilt symptoms and soft rot symptoms were collected from the farmer’s field. The fungal pathogen Pythium aphanidermatum and the bacterial pathogen, Ralstonia solanacearum were isolated from the rhizomes showing typical symptoms, and pure cultures were maintained. Since Pleurotus florida and P. sajor-caju are the most suitable mushroom species in Kerala, they were selected for the production of spent mushroom substrate. The substrate used were agricultural waste materials like paddy straw, saw dust and neopeat. The enumeration of microorganisms at different stages of mushroom growth were made and the fungi commonly observed were Aspergillus sp. (FA-2 and FA-3) and Trichoderma sp. (FA-1). Maximum number of microbial population was noticed in paddy straw, whereas it was minimum in neopeat. From four different stages of mushroom growth, five different types of bacteria, BA-1, BA-2, BA-3, BA-4 and BA-5 were observed. An in vitro evaluation study was conducted to find out the efficiency of isolated organisms against the pathogens P. aphanidermatum and R. solanacearum. Among the bacterial isolates tested against P. aphanidermatum, BA-4 and BA-5 were effective, whereas against R. solanacearum, only BA-5 was found effective. All the three fungal isolates, FA-1, FA-2 and FA-3 gave 100 per cent inhibition against both the pathogens. But FA-1 was the most effective one. These five antagonists were selected for the compatibility studies by dual culture technique. The selected fungal antagonist FA-1 was identified as Trichoderma viride by studying cultural and morphological characters. The bacterial isolates BA-4 and BA-5 were identified as Pseudomonas aeruginosa and Klebsiella pneumoniae respectively. The effect of spent mushroom substrate for the management of rhizome rot complex disease of ginger was assessed under pot culture conditions. Two separate experiments were conducted for the management of P. aphanidermatum and R. solanacearum. All the treatments were applied at three times- at the time of planting, 60 DAP and 120 DAP. Among the various treatments for the management of both the pathogens associated with rhizome rot complex disease, T. viride from SMS and paddy straw SMS of P. sajor-caju as mulch were found to be the best giving cent per cent disease control. In control treatment, cent per cent disease incidence was noticed. In the experiment for the management of P. aphanidermatum, hundred per cent germination was noticed in the treatment, T. viride from SMS + Reference culture T. viride. Among the treatments with SMS as mulch, paddy straw SMS of P. sajor-caju showed better germination percentage. But in the experiment for the management of R. solanacearum, the treatment Klebsiella pneumoniae showed 100 per cent germination followed by the treatment T. viride from SMS. The growth parameters like number of tillers, number of leaves per tiller and height of tillers were highest in the treatment T. viride from SMS, at two, three, four and five months of planting, in the experiment for the management of P. aphanidermatum. But in the experiment for the management of R. solanacearum, the treatment paddy straw SMS of P. sajor-caju as mulch showed highest number of tillers. Number of leaves per tiller was highest in the treatment K. pneumoniae + reference culture P. fluorescens, in the first three months and the treatment with paddy straw SMS of P. sajor-caju as mulch for the next two months. Height of tillers was observed maximum in the treatments applied with T. viride from SMS. The same trend was noticed in the case of yield also. In the experiment for the management of P. aphanidermatum, highest yield was observed in the treatment T. viride from SMS. But in the experiment for the management of R. solanacearum, the treatment paddy straw SMS of P. sajor-caju as mulch produced highest yield. SMS is rich in microflora with antagonistic effect against pathogenic microorganisms associated with ginger plants. From the pot culture experiment it was clear that for the management of R. solanacearum and plant growth promotion, best treatment is paddy straw SMS of P. sajor-caju as mulch. High cellulolytic capacity of P. sajor-caju favour the maximum degradation of the substrate, thereby provides a niche for the multiplication of favourable microorganisms with antagonistic effect. Whereas, best treatment for the management of P. aphanidermatum and plant growth enhancement is T. viride from SMS. It was found to be better than reference culture T. viride. In addition the beneficial microorganisms may be having plant growth promoting activity which contributes to increased yield in the various treatments. So from the present study, it can be concluded that the use of paddy straw SMS of P. sajor-caju as mulch, along with the application of T. viride from SMS can be considered as an effective management practice against the rhizome rot complex disease of ginger. Depending on the location and climatic condition in which the mushroom crop is raised, the microorganisms harbouring in SMS will vary and they can be used for disease management in various crops.ThesisItem Open Access Cataloguing and management of major diseases of monopodial orchids(Department of Plant Pathology, College of Horticulture,Vellanikkara, 2012) Meera, T M; KAU; Vimi, LouisDisease is one of the major production constraints in orchid cultivation. Hence an investigation has been undertaken to study the symptomatology, etiology and management of various diseases of important monopododial orchids viz., Phalaenopsis, Vanda (Basket Vanda), Mokara and Arachnis. A survey was conducted in different locations of Thrissur District viz., orchidarium of AICRP on Floriculture Improvement, Department of Pomolgy and Floriculture, COH, Vellanikkara, nurseries at Cheroor, Thrissur, Perinjanam, Madakkathara and Kanimangalam. Different fungal and bacterial diseases were observed and isolation of pathogen yielded eleven fungal pathogens and two bacterial pathogens. The pathogenicity of these organisms was proved by artificial inoculation.Symptomatology of various diseases was studied in detail under natural and artificial conditions. Fusarium sp. caused wilt symptom in Phalaenopsis and caused leaf spot in Arachnis. Sclerotium sp. caused collar rot in Phalaenopsis and dry rotting in Basket Vanda, Anthracnose pathogen, Colletotrichum sp. produced different symptoms in Phalaenopsis, Mokara and Arachnis. In Phalaenopsis, the symptom was irregular sunken spot, in Mokara leaf blighting and in Arachnis, round sunken spot. Heart rot was the symptom of Phytophthora infection in Basket Vanda. Botryodiplodia leaf spot of Basket Vanda was characterized by greyish white coloured spot with black margin and pycnidia at the centre. Alternaria leaf spot of Mokara, was round to oval shaped with a vertical splitting through the centre of the spot. Soft rot of Phalaenopsis by Erwinia sp. and bacterial wilt of Basket Vanda by Burkholderia sp. were the bacterial diseases observed during the survey. Water soaking and rotting of leaves were the symptoms of soft rot while yellowing, wilting and leaf detachment were the symptoms of bacterial wilt.Cultural characters and morphological characters of fungal pathogens were studied and they were identified at species level. Cultural, morphological and gram reaction of bacterial pathogens were studied and they were identified at species level by molecular techniques.For the management of fungal pathogens, an in vitro evaluation was conducted with fungicides and liquid formulation of Pseudomonas fluorescens. Most of the fungicides revealed cent per cent inhibition on the growth where as P. fluorescens showed 53 - 86 per cent inhibition. For the management of bacterial pathogens, an in vitro evaluation was conducted with cowdung, liquid formulation of P. fluorescens, cowdung + P. fluorescens and Streptocycline and differences were observed in their inhibitory properties. Seasonal influence on the incidence of diseases of monopodial orchids was studied for one year. Influence of temperature, humidity and rainfall was prominent in the incidence of Fusarium wilt, collar rot and soft rot of Phalaenopsis whereas not very prominent in the incidence and severity of Botryodiplodia, Alternaria and Fusarium leaf spot diseases of Basket Vanda, Mokara and Arachnis respectively . Incidence of Fusarium wilt was more in the month of November, collar rot in the month of December whereas bacterial soft rot was prominent in rainy months.ThesisItem Open Access Serodiagnosis and standardization of techniques for production of virus free planting materials of Cassava variety,Vellayani Hraswa(College of Agriculture, Vellayani, 2012) Asha, B Nair; KAU; Umamaheswaran, KCassava (Manihot esculenta Crantz), the staple or subsidiary food for about one fifth of the world’s population, is an important source of dietary calories. Among the diseases and pests of cassava, cassava mosaic disease (CMD) is a serious factor limiting the productivity of cassava and the variety, Vellayani Hraswa is found to be highly susceptible to the disease. CMD is caused by viruses belonging to the genus Begomovirus of the family Geminiviridae. Since the crop is vegetatively propagated, the CMD is carried from one crop cycle to the next, through the use of infected cuttings used as planting material. Studies were conducted for the diagnosis of cassava mosaic geminivirus and production of disease free planting material of cassava variety, Vellayani Hraswa causing the mosaic disease of cassava in Kerala. The characteristic symptoms observed include chlorotic areas on the leaf and bright mosaic pattern of yellow or pale green patches. In severely affected plants, leaf distortion and twisting, reduction in size of leaf lamina and stunting of plant growth were observed. Host range studies were conducted and the virus was found to infect only Datura stramonium belonging to the Solanaceae family. The sap transmission of the virus using 0.1 M sodium phosphate buffer (pH 7.0) was found not successful from cassava to cassava. Chlorotic lesions were produced on the local lesion host, Datura which later turned necrotic. Insect transmission studies revealed that only whiteflies (Bemisia tabaci) were able to transmit the virus whereas the spiralling whiteflies (Aleurodicus dispersus) and mealy bugs (Ferrisia virgata) associated with cassava could not transmit the virus. Graft transmission of the virus was also found successful. The pathophysiological studies revealed that the total carbohydrate and phenol contents in graft inoculated plants were significantly higher compared to the uninoculated cassava plants. The content of chlorophyll a, b and total chlorophyll were lower in inoculated cassava plants. There was a decrease in total protein content in inoculated plants compared to the healthy plants but was found to be increasing at different stages of inoculation. The activity of the defense related enzymes were higher in case of inoculated plants than that of the control plants. Protein profile study of virus infected cassava plants using SDS- PAGE showed an extra novel protein with approximate molecular weight of 40 KDa corresponding to the coat protein of the virus. Isozyme analysis of polyphenol oxidase produced three isoforms in both healthy and inoculated plants with relative mobility (Rm) values of 0.68, 0.75 and 1. Two isoforms with Rm values 0.63 and 0.74 were observed in case of peroxidase isozyme analysis in inoculated plants whereas only one in healthy plants. The activities of the isoforms were more in the inoculated plants. Serological and molecular characterization of the virus was also done. The virus was partially purified from the infected leaves and antiserum with a titre of 1: 512 was produced. Serological characterization of the virus using ELISA showed the close relationship of ACMV and ICMV with the disease. Presence of the virus in the whitefly vector B. tabaci was confirmed through DAC- ELISA. Detection of the virus was also done using Dot Immunobinding Assay (DIBA). A field level diagnostic kit using DIBA was developed. Multiplex PCR using ICMV and SLCMV specific primers produced an amplicon of size 600 bp corresponding to the DNA- A fragment of SLCMV. Sequencing of the PCR product obtained 584 bp long nucleotide sequences. The cluster dendrogram constructed by multiple alignment of sequences showed three major clusters and the isolated viral sequence shared the same cluster with SLCMV isolates collected from Kerala. The meristem from the infected cassava plants were regenerated into complete plantlets within 30 days in a suitable MS media supplemented with benzyladenine (0.5 ml l-1), naphthalene acetic acid (0.1 ml l-1) and gibberellic acid (0.1 ml l-1) of millimolar concentration. Root induction was better in the basal MS media and was tested for the presence of the virus by subjecting it to DAC- ELISA. The absorbance of the plantlet regenerated from healthy and infected meristem were found to be 0.22 and 0.31 respectively which was on par with the healthy field sample (0.28) but much lower than that of the infected field sample (1.23) which was used as the positive control. The virus free plantlets were transferred to pots containing sand, soil and compost in 1:1:1 ratio and were grown in a green house. Meristem culture and virus indexing by use of protein and nucleic acid based detection thus, can be used as a viable strategy for the early detection and elimination of the virus and hence for the production of virus free planting materials.ThesisItem Open Access Bio intensive management of blast disease of rice (Oryza sativa L.)(Department of Plant Pathology, College of Agriculture, Vellayani, 2012) Yamini, Varma C.K; KAU; SanthakumariThe present study on ‘Bio intensive Management of Blast Disease of Rice (Oryza sativa L.)’ was conducted at College of Agriculture, Vellayani and Regional Agricultural Research Station, Pattambi during 2008-2010. A survey conducted during kharif and rabi seasons 2008, in six panchayats from three different blocks, in Palakkad district, revealed that maximum disease incidence was in Pattithara panchayat during rabi and maximum disease severity was in Chalissery panchayat during kharif. The disease intensity and severity were high during kharif season, than rabi and in all the panchayats disease incidence occurred, 60 days after sowing. The fungus produced symptoms on leaves and neck of panicle. Sixty isolates from leaf samples were obtained from the six panchayats which proved the Koch’s postulates. After preliminary screening, thirteen isolates out of the 60 were selected. Out of the thirteen isolates, five isolates, P2, P4, P5, P9 and P12 had high virulence and these were selected for further studies. Among the five isolates the most virulent isolates like P4, P2 and P12 were having dark black coloured colonies with good growth. The isolate from Chalissery (P4) which was highly virulent, with maximum radial growth, mycelial dry weight, conidial size, and sporulating nature was selected for further studies. The isolate was tentatively identified as Pyricularia oryzae and further confirmed at IARI, New Delhi with accession number, as ITCC-7019. Among the media tested Potato Dextrose Agar was best followed by Oat Meal Agar. Among the liquid media, the Potato Dextrose broth supported maximum growth of Pyricularia. The optimum range of temperature for growth of Pyricularia was 25 – 30 °C, and pH value for maximum growth was 6.5. The most potent fungal antagonist Trichoderma sp. was obtained from Kadambazhippuram panchayat and the bacterial antagonist fluorescent pseudomonad was obtained from Thrithala panchayat. The per cent of inhibition was highest for Trichoderma sp. under in vitro conditions and hence this culture was selected as the fungal antagonist for further studies. The hyphae of the Trichoderma were found to coil around the hyphae of blast pathogen and in some areas disintegration of pathogen hyphae were also observed. Based on the morphological characteristics of the fungal antagonist the isolate was tentatively identified as Trichoderma harzianum and further confirmed at IARI, New Delhi, with accession number, as ITCC-7018. The fluorescent pseudomonad with highest percentage of inhibition was selected as bacterial antagonist for further studies. Based on the morphological, cultural, biochemical and physiological characteristics, the bacterial antagonist has been tentatively identified as Pseudomonas fluorescens biovar - 1. Among the plant products, the percentage of inhibition on the growth and spore germination of Pyricularia oryzae was significantly higher for the Palmarosa oil @ 0.5ml/ l) under in vitro conditions. Neemazal @ 5ml/ l was the second best plant product. The percentage reduction of spore germination was also highest for Palmarosa oil followed by Neemazal. Carbendazim (1.5g/ l), followed by Propiconazole (1ml/ l) were the best performing fungicides under in vitro conditions and they were selected as the best two chemical fungicides for further studies. Two blasticides viz., Isoprothiolane (1.5ml/ l) and Carpropamid (1ml/ l) were also selected as additional treatments. Salicylic acid (0.1ml/ l) and Benzoic acid (0.1ml/ l) were selected as best two resistance inducers from the pot culture experiment – I, since they reduced the disease severity significantly and increased the yield. In pot culture experiment-II, plants treated with Palmarosa oil + Carbendazim (T23) had significantly reduced the blast disease. Next best performance was when Salicylic acid combined with Pseudomonas fluorescens or Trichoderma. The two biocontrol agents, Pseudomonas fluorescens and Trichoderma were effective when individually combined with plant products like, Palmarosa oil, or Neemazal, or resistance inducer, Salicylic acid. The biometric observations like, height of the plant, number of total and productive tillers, grain yield and straw yield were also considerably higher for the above treatments. The best nine treatments with lowest PDI and highest yield from this experiment were selected for field experiments. Palmarosa oil and Neemazal was compatible with Trichoderma, but the systemic fungicide, Carbendazim inhibited growth of Trichoderma followed by Propiconazole. Pseudomonas fluorescens was compatible with the two plant products, Palmarosa oil and Neemazal as well as two fungicides, Carbendazim and Propiconazole. It was also found that T. harzianum and Pseudomonas fluorescens were compatible to each other. Field Experiment-I showed that 75 DAT, T23 (Palmarosa oil + Carbendazim) had significantly lowest percentage of disease incidence and this was on par with T21 (Palmarosa oil + Salicylic acid), T36 (Trichoderma + Pseudomonas fluorescens + Salicylic acid) and T34 (Trichoderma + Pseudomonas fluorescens + Palmarosa oil). The treatment T23 (Palmarosa oil + Carbendazim) had the highest effect on reducing the PDI, 75DAT along with four treatments viz., T21, T27, T36 and T34 which were on par in their effects. The treatment T23 (Palmarosa oil + Carbendazim), produced significantly more height, higher number of total tillers, and productive tillers. Significantly higher grain yield /ha was obtained for T23 and T34. Significantly maximum straw yield /ha was also obtained for T23 In the diseased plants there was a steady decrease in chlorophyll ‘a’, ‘b’ and total chlorophyll. But in the uninoculated healthy plants, chlorophyll content remained same throughout the observation period. The activity of defense related enzymes viz., peroxidase, poly phenyl oxidase and phenyl alanine ammonia lyase activity, was significantly higher in rice plants on 5th day after inoculation for the treatment Trichoderma + Pseudomonas fluorescens + Palmarosa oil followed by Trichoderma + Pseudomonas fluorescens + Salicylic acid. Enzyme activities were least in the un inoculated plants. In the second experiment (kharif 2010) 75DAT, T23 (Palmarosa oil + Carbendazim) had significantly lower percentage of disease incidence. Two additional blasticides were also on par with T23 The biometric observations also followed the same trend. Significantly maximum grain yield and straw yield /ha was obtained for T23. The pooled analysis during two succeeding years, (kharif 2009 and 2010) revealed that, T23 had the lowest PDI with highest efficiency in reducing the blast disease. Four treatments viz., T21, T27, T36 and T34 which were on par in their effects, with T23. The B:C ratio was highest for T23 (Palmarosa oil + Carbendazim) followed by T21 (Palmarosa oil+ Salicylic acid), T27 (Neemazal + Carbendazim) (2.5), T34 (Trichoderma + Pseudomonas fluorescens + Palmarosa oil), T36 (Trichoderma + Pseudomonas fluorescens +Salicylic acid) and fungicidal check T41 (Tricyclazole). Exactly the same value of the maximum B:C ratio was obtained for Isoprothiolane. The relationship between blast disease severity and weather factors during kharif 2010, indicated positive correlation between disease severity and maximum temperature, relative humidity, rainfall, and number of rainy days but negative correlation with minimum temperature. The outcome of this research work which can be recommended in the rice organic farming techniques, are the following 1. Trichoderma + Pseudomonas fluorescens + Palmarosa oil 2. Trichoderma + Pseudomonas fluorescens +Salicylic acid 3. Palmarosa oil + Salicylic acid The result of molecular diagnosis of five selected cultures of blast pathogen, based on the D1/D2 regions in PCR analysis showed 99% similarities for 4 cultures (P2, P4, P9 and P12) and 97% similarities in one culture (P5) with the Magnaporthe oryzae, which accurately proved the identity of blast pathogen.