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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, Louis
Bitter 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.
Open Access
Molecular characterization, host range and integrated management of bhindi yellow vein mosaic disease
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2019) Chinju, E A; KAU; Anita Cherian, K
Bhindi (Abelmoschus esculentus (L.) Moench) is one of the most important vegetable crops cultivated across the globe. However, its cultivation is often hindered by biotic stresses like incidence of pests and diseases. Among the diseases, yellow vein mosaic disease is one of the major constraints in bhindi cultivation which leads to 100 per cent yield loss especially when infected at an early stage of the crop. In recent years, the evolution of new viral strains is a serious problem especially among Begomoviruses belonging to the family Geminiviridae which has an adverse effect on the host plant resistance. Considering these facts, the present study was undertaken to carry out the molecular characterization of the virus causing bhindi yellow vein mosaic disease (BYVMD), to study the host range and seed transmission of the virus and to develop a sustainable disease management strategy. The project was initiated with purposive sampling survey conducted in elevan different locations of Thrissur district, Kerala. The disease incidence recorded during the survey ranged from 61.20 to 98.16 per cent while the disease severity ranged from 48 to 90 per cent. The predominant symptoms observed on the leaves of infected plants under natural conditions were vein clearing, vein thickening, reduction in leaf area, bleached appearance and marginal necrosis. A novel type of symptom observed during the survey was general yellowing of leaves with severe puckering along the veins and upward curling of leaves. Linear cholorotic striations were observed on the calyx of the flower buds. The immature fruits produced by the infected plants showed linear chlorotic striations, while the mature fruits were bleached in appearance along with reduction in fruit size. The plants infected during the early vegetative stage were extremely stunted. The major symptoms developed under artificial conditions were vein clearing, vein thickening and puckering of leaves. xxiv Histopathological studies of the infected leaf revealed disruption of parenchymtous cells in the epidermis, disintegration of chloroplast, reduction in number of metaxylem and protoxylem along with abnormality of phloem vessels. The studies on virus transmission confirmed that it is transmitted through grafting and insect vector, Bemisia tabaci. The presence of virus inside the insect body was also confirmed through polymerase chain reaction (PCR) based molecular technique. The studies on seed transmission revealed that BYVMD is not seed-borne. Host range studies revealed that weed species Synedrella nodiflora and Hemidesmus indicus were proved to be hosts of the begomovirus. Molecular detection of the virus causing BYVMD was standardized through PCR, using universal primer specific to the core coat protein gene of Begomovirus which yielded amplicons at expected size of about 550 bp. The amplification was also carried out using primers specific to coat protein gene of bhindi yellow vein mosaic virus (BYVMV) and okra enation leaf curl virus (OELCuV) which yielded amplicons at expected band size of about 770 bp. The molecular characterization of the elevan isolates was carried out through in silico analysis to identify the virus associated with BYVMD and for diversity analysis. The results revealed that all the isolates showed 99-100 per cent nucleotide homology to OELCuV. BLASTp analysis of the isolates also showed 100 per cent identity with coat protein of OELCuV and thus confirming that the virus causing yellow vein mosaic disease in bhindi in Kerala is okra enation leaf curl virus. The identity was further confirmed through DNA barcoding technique and species demarcation analysis. A field experiment was also conducted to develop a disease management package against BYVMD. Among the seven treatments, T5 i.e., integrated management with early seedling protection using insect proof net + yellow sticky trap + seed bio-priming and foliar spray of PGPR mix II + alternate foliar spray of Bougainvillea spectabilis and azadiractin was found to be most effective with xxv lowest disease incidence and severity, least whitefly population and maximum yield. It is concluded that yellow vein mosaic disease affecting bhindi cultivation in Kerala is caused by okra enation leaf curl virus, an evolved strain of BYVMV. This virus is transmitted through grafting and the insect vector Bemisia tabaci and not though seeds. The outcome of the study would also facilitate early detection and elimination of sources of infection so as to reduce the spread of the disease. An integrated disease management package was also developed for the benefit of farming community.
Open Access
Characterization and evaluation of Pseudomonas spp. for abiotic stress tolerance
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2019) Reshma, K S; KAU; Reshmy, Vijayaraghavan
Pseudomonas spp. are one among the most extensively used biocontrol agent in plant disease management to control foliar, soil borne or seed borne pathogens. However, prevalence of abiotic stresses such as drought, high temperature, salinity and acidity may affect the field performance due to poor survival under adverse conditions. To date, very little effort has been taken to tap microbial diversity from stressed ecosystems of Kerala. Thus, a study was undertaken to isolate native strains of Pseudomonas spp. having inherent stress tolerance. Purposive soil sampling survey was conducted during the period February- April 2018 and a total of 26 representative soil samples were collected from four districts viz., Ernakulam, Palakkad, Thrissur and Alappuzha. It was revealed that various locations of Palakkad and Thrissur district experienced high temperature (40-46oC) and low moisture of 1.1- 3.9 per cent. Samples procured from Alappuzha and Ernakulam districts were extremely acidic (pH 3.4- 4.5) andsaline (EC 4.1-6.68 dSm-1). The results of soil analysis confirmed that the collected soil samples were abiotically stressed and there could be chances of obtaining stress tolerant native isolates of Pseudomonas spp. For isolation of Pseudomonas spp., soil samples weresubjected to serial dilution and plating technique in King’s B agar and Pseudomonas agar base, among which King’s B agar yielded optimum number of Pseudomonas colonies at 105 dilution.Maximum population of Pseudomonas spp. was recorded in samples procured from Moncombu (1.3-7.8x106cfu g-1), whereas, minimum population was observed in soil samples collected from Vyttila (3.14- 6.01 x 104cfu g-1). A total of 15 isolateswere purified and designated with sample codes based on the place of collection. Among the nine fluorescent isolates, M7 and P7 were identified as Pseudomonas aeruginosa, an opportunistic human pathogen based on its growth at 42oC and on King’s A agar medium and thus, were eliminated. Isolates of Pseudomonas spp. were subjected to in vitro screening for abiotic stress tolerance and compared with reference culture of KAU. The isolates P2, M4 and T5 were selected as temperature tolerant (50oC), M4 and V4 as salt tolerant (1.5 M NaCl), P4 and T4 as xxxii drought tolerant (30% PEG) and M4 and M5 as acid tolerant (pH 3.5) strains. Subsequently, a total of seven abioticstress tolerant isolates viz., M4, M5, P2, P4, V4, T4 and T5 were evaluated further for their in vitro antagonistic potential against five major soil borne fungal pathogens using Bangle method.The four isolatesviz., P2, P4, M4 and M5 were selected as the potential antagonistic strains since they outperformed reference culture of KAU and showed higher and consistent biocontrol activity with per cent growth inhibition ranging from 62.21 to 91.00 per cent against Phytophthora capsici, Pythium aphanidermatum, Sclerotium rolfsii, Rhizoctonia solani and Fusarium oxysporum. Further studies revealed M4, M5 as better producers of ACC deaminase enzyme and M4, P4 as better producers of exopolysaccharide. Highest cellulase and β-1, 3 glucanase production was recorded by the isolates P2 and M4 respectively. A preliminary screening of the isolates were carried out for testing the bioefficacy on cowpea seeds against Rhizoctonia solani using roll towel method. The results revealed that the isolate M4 was highly efficient, reference culture as efficient and the other isolates P2, P4 and M5 as moderately efficient.All the four isolates were further evaluated for their in vivo activity through a pot culture experiment using cowpea- R. solani system. Eventhough, M4 outperformed all other isolates with highest seed germination, biometric characters and yield as well as with lowest per cent disease incidence, other three isolates were also found superior or equally efficient as reference culture of KAU. The best performing isolates with promising traits of stress tolerance, antagonism and plant growth promotion viz., P2, P4, M4 and M5 were identified based on cultural, morphological, biochemical and molecular characterization. The isolate P2 was identified as Pseudomonas putida, P4 as P. fluorescens and M4 and M5 as P. aeruginosa. Thus, the current investigation has thrown light on the prevalence of abiotic stress tolerant strains of Pseudomonas spp. in stressed ecosystems which would significantly help the farming community to overcome such drastic climate changes. However, the study should further be complemented with large scale multilocational trials to prove their efficacy under field conditions.
Open Access
Characterisation of Ralstonia solanacearum (Smith) Yabuuchi et al. infecting solanaceous vegetables in relation to Physico-chemical and Biological properties of soil
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2019) Anjali, V A; KAU; Sainamole Kurian, P
Ralstonia solanacearum, the causal agent of vascular wilt disease of crop plants has been ranked as the second most important bacterial pathogen in the world next to Pseudomonas syringae. The high diversity exhibited by the pathogen hampers the breeding for resistance, consequently the resistant varieties developed may not express uniform level of resistance in different geographical locations. Being a soil inhabitant, the survival of R. solanacearum is influenced by physico-chemical and biological properties of soil. Considering these facts, present investigation was carried out with the objective of characterisation of R. solanacearum from different agro ecological units (AEUs) of Kerala and to determine the effect of soil physical, chemical and biological properties on the pathogen. Purposive sampling survey was conducted in four AEUs of Kerala viz., North Central laterite (NCL), Marayur hills (MH), Southern laterite (SL) and Palakkad central plains (PCP) from March to September 2018. Two locations from each AEUs were selected for the survey. The per cent incidence in different locations ranged from 20 to 88 per cent. The pathogen was isolated from infected plant samples collected during the survey on triphenyl tetrazolium chloride (TZC) agar and pathogenicity was proved by inoculation on respective hosts. The rhizosphere soil samples of healthy and diseased plants were collected from each location. The population of the pathogen present in the soil was quantified and it ranged from 20.66 x 104 cfu g-1of soil to 137.66 x 104 cfu g-1 of soil. A strong positive correlation was observed between pathogen density in soil and per cent disease incidence (PDI). A total of eight isolates were collected, purified and maintained for all the experiments. The colony characters of different isolates on TZC agar showed considerable variation and the morphology of the bacterial cells was studied using scanning electron microscopy. Typical rod shaped cells with size 0.3-0.5 µm x 1.2-1.7 µm were observed. The molecular characterization of the isolates was done by PCR amplification and sequencing of 16S rRNA. The sequences were subjected to in-silico analysis which confirmed the identity of all isolates as R. solanacearum (Smith) Yabuuchi et al. The phylogenetic analysis revealed that the eight isolates collected from different AEUs clustered on different branches of the tree while those from the same AEUs clustered together. This indicates considerable variation among the isolates in accordance with location which can be attributed to the difference soil parameters in these locations. The isolates of the pathogen were further categorized into races and biovars based on pathogenicity on differential hosts and utilization of disaccharides and hexose-alcohols respectively. The results revealed that two isolates from Marayur hills (MH 1 and MH 2) belong to race 3, biovar II whereas two from Palakkad central plains (PCP 1 and PCP 2) belong to race 1 biovar III A. The other four isolates collected from Northern central laterite (NCL 1 and NCL 2) and Southern laterite (SL 1 and SL 2) were identified as race 1, biovar III. The physico-chemical and biological properties of the soil samples collected during the survey were analysed using standard protocols. The statistical analysis using paired sample t-test revealed significantly higher soil pH, organic carbon, available K, Ca and Fe content and soil microflora in rhizosphere soil of healthy plant compared to diseased. A significant positive correlation was observed between PDI and soil parameters viz., water holding capacity and bulk density whereas soil pH and available Ca content exhibited a negative correlation with PDI. A similar trend was observed in the case of pathogen population also. Further, multiple regression analysis was performed to assess the extent of variation contributed by different soil parameters on PDI and pathogen population. The results indicate that 96.8 per cent variation in the bacterial wilt incidence is explained by soil pH and available Ca content in the rhizosphere soil with negative correlation whereas bulk density and Ca content contributed 92.2 per cent in the build-up of population of Ralstonia solanacearum in soil. The study revealed the influence of soil factors on bacterial wilt disease incidence, population of R. solanacearum and pathogen variability. Hence, it is concluded that, manipulation of soil factors can play a major role in integrated management of the disease. Furthermore, the variability of pathogen according to geographical region, may be considered while planning resistance breeding programmes against bacterial wilt disease.
Open Access
Physiological and cultural studies on blue oyster mushroom (Hypsizygus ulmarius (Bull.:Fr.) Readhead)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2016) Sumi, I; KAU; Geetha, D
The present study entitled “Physiological and cultural studies on blue oyster mushroom (Hypsizygus ulmarius (Bull.:Fr.) Redhead)” was carried out in the mushroom unit, Instructional Farm, College of Agriculture, Vellayani during 2014-2016, with the objective to standardize the technology for cultivation of Hypsizygus ulmarius and to study its morphological and physiological aspects. The initial culture of H. ulmarius was isolated from the mushroom beds maintained in the mushroom unit of instructional farm through tissue culture method and purified by hyphal tip method. Morphological studies of H. ulmarius showed that the sporocarps were medium to large in size having a dark blue colour in the pinhead stage which became creamy white on maturity with an irregularly shaped, convex pileus with gills attached to the stem, but not decurrent and cylindrical, smooth and eccentric stipe. Microscopic studies revealed septate hypahe with clamp connection, oval shaped, hyaline basidiospores and the spore print was white. Studies on developmental morphology showed that H. ulmarius took an average of five days from the day of pinhead formation to complete maturity. The maximum mycelial growth was recorded on potato dextrose agar. A temperature of 25 0C, pH of 8 and dark conditions are found favourable for maximum mycelial growth. Evaluation of different substrates for spawn production revealed that paddy grains was the best medium in which spawn run was completed in fifteen days with thick fluffy growth and recorded less contaminants followed by wheat and sorghum. Evaluation of different substrates for mushroom production revealed that paddy straw was the best material for the cultivation of blue oyster with a total yield of 985 g kg-1 from three harvests followed by rubber sawdust (905 g kg-1). The minimum time for mushroom production was recorded for sugarcane bagasse and the maximum time for rubber sawdust. The average weight of sporocarp was maximum in mushrooms harvested from rubber sawdust and the maximum number of sporocarps was recorded in paddystraw. Beds prepared from sugarcane bagasse were heavily contaminated with Trichoderma sp. When compared with Pleurotus florida, H. ulmarius took more time (18 days) for complete spawn run in paddy grains and the yield was higher on paddy straw (1.096 kg kg-1) than P. florida (976 g kg-1). Infestation of pests viz., phorid flies (Megaselia sp.) and staphylinid beetles were prevalent during spawn run as well as sporocarp formation. The competitor moulds recorded were Trichoderma sp., Aspergillus sp. and Coprinus sp. Analysis for the proximate constituents in H. ulmarius revealed that it contains appreciable amount of carbohydrate (29 %), protein (32 %) and fibre (17.69 %). Sensory evaluation was done on steam cooked mushrooms for attributes like appearance, colour, texture, flavor and taste using five point score card and an overall acceptability score of 3.6 was obtained for H. ulmarius compared to P. florida (3.0). In the preference study conducted for both the mushrooms using Hedonic rating scale, 30 per cent of evaluators extremely liked H. ulmarius than P. florida (10 %). The study on the keeping quality of mushrooms in normal atmospheric condition indicated a shelf life of eight hours for H. ulmarius compared to six hours for P. florida. The study also showed that blue oyster mushrooms stored under refrigeration (4 0C) in perforated polythene covers had better shelf life (5 days) compared to P. florida (3 days). The present study indicated that blue oyster mushroom can be cultivated successfully in tropical areas on locally available materials like paddy straw and rubber saw dust under favourable climatic conditions viz., 26-28 0C temperature, more than 90 per cent relative humidity and good aeration. The variety is superior to the presently growing oyster mushroom (P. florida) in terms of yield, presence of appreciable amount of proximate constituents and keeping quality.
Open Access
Integrated management of rhizoctonia leaf blight of amaranthus (Amaranthus tricolor L.)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2016) Gireesh; KAU; Radhakrishnan, N V
The study entitled “Integrated management of Rhizoctonia leaf blight of Amaranthus (Amaranthus tricolor L.)” was conducted at the College of Agriculture, Vellayani and Coconut Research Station, Balaramapuram during 2014-2016 with the objective to investigate the effect of soil solarization, biocontrol agents, chemical activator, indigenous formulations and new generation fungicides on growth, yield and severity of foliar blight of amaranthus. Samples of the infected leaves showing Rhizoctonia leaf blight in amaranthus were collected from Vellayani, Kalliyoor, Venganoor and Kakkamoola locations. Among the four isolates of the pathogen, the Vellayani isolate gave significantly superior growth rate with minimum of six days for sclerotial formation. Koch‟s postulates were proved for the pathogenicity of different isolates of Rhizoctonia solani. All the four isolates have taken three days for the first symptom development but the progression of lesion size of Vellayani isolate was maximum compared to all other isolates, hence the Vellayani isolate was selected as the most virulent isolate for use in further in vitro studies. Evaluation of biocontrol agents for in vitro suppression of R. solani showed that Trichoderma harzianum completely overgrown the pathogen with maximum inhibition of 49.56 % compared to Pseudomonas fluorescens (28.30 %). Under in vitro evaluation of chemical activator, different concentrations of Acibenzolar-S- Methyl (ASM) against pathogen, 100 ppm concentration recorded the maximum mycelial inhibition of 75.67 % and 5 ppm concentration recorded the minimum mycelial inhibition of 27.70 %. Among indigenous organic formulations, turmeric powder and baking soda combination inhibited the maximum growth of the pathogen by 64.40 %. In the in vitro studies with new generation fungicides,mancozeb in cow dung supernatant (0.4 %) and tebuconazole (0.1 %) recorded the 100 % mycelial inhibition of the pathogen. Field studies on disease suppression and plant growth promotion was carried out as two experiments, one in soil solarized plots and the other in non solarized plots. Soil solarization along with soil application of ASM (75 ppm) and foliar application of ASM (100 ppm) recorded the lowest disease incidence of 30.41 % and 30.42 % respectively, which was superior when compared with foliar application of ASM (100 ppm) and soil application of ASM (75 ppm) with the disease incidence of 37.06 % and 38.84 %. Soil solarization + foliar spray of tebuconazole (0.1 %) recorded the minimum disease index of 37.85 % which was superior compared to foliar spray of tebuconazole (0.1 %) with the disease index of 39.28 %. Among the biocontrol agents soil solarization + foliar spray of Pseudomonas fluorescens (2 %) gave minimum disease index of 45.22 % which was greater compared to foliar spray of P. fluorescens (2 %) with the disease index of 51.66 %. In case of indigenous organic formulations, soil solarization + foliar spray of fish amino acid (5 %) given the maximum control of the disease with the disease index of 49.51 % which was superior to foliar spray of fish amino acid (5 %) with disease index of 63.59 %. The number of days taken for flowering in soil solarized plots ranged from 28.67 to 35 days where as the number of days taken for the flowering of amaranthus in non solarized plots was ranged from 27.27 to 31.67 days. At the time of harvest, soil solarization + mancozeb in cow dung supernatant (0.4 %) recorded maximum plant height of 127.07 cm which was higher compared to foliar spray of azoxystrobin (0.15 %) with plant height of 117.60 cm. Maximum of 78.00 number of leaves were recorded by soil solarization + foliar spray of azoxystrobin (0.15 %) which was greater compared to foliar spray of azoxystrobin (0.15 %) with 67.67 number of leaves.Soil solarization + foliar spray of azoxystrobin (0.15 %) gave the highest yield in terms of fresh weight by 26975.00 kg/ha and dry weight of 4233.33 kg/ha which was superior when compared with foliar spray of tebuconazole (0.1 %) with the fresh weight of 23375.00 kg/ha and dry weight of 3362.50 kg/ha. It is concluded that soil solarization for 31 days with the foliar application of tebuconazole (0.1%) can effectively control the Rhizoctonia leaf blight disease severity with plant growth and yield promotion under field conditions.
Open Access
Molecular detection and characterization of phytoplasma infecting brinjal (solanum melongena L.)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2015) Saranya, S S; KAU; Umamaheswaran, K
The study entitled “Molecular detection and characterization of phytoplasma infecting Brinjal (Solanum melongena L.) was conducted at the Department of Plant Pathology, College of Agriculture, Vellayani, with the objectives to study the symptom development, transmission, molecular detection and characterization of phytoplasma infecting brinjal and its relationship with phytoplasma diseases of other crop plants. Brinjal little leaf (BLL), collected from the Crop museum, College of Agriculture, Vellayani and catharanthus little leaf (CLL) obtained from Coimbatore were maintained for further studies. Symptomatology revealed the characteristic little, narrow, soft, glabrous and smooth leaves produced as clusters along with yellowing, proliferation of axillary shoots, shortened internodes, stunted bushy or rosette appearance and phyllody, the conversion of floral parts into leaf like structures. The graft transmission was found to be 100% successful while the percentage transmission by dodder was only 10% in brinjal and 20% in catharanthus. Phytoplasma was maintained in vivo in plants by grafting and in vitro by culturing the infected explants on MS media supplemented with 0.2 mg l-1 BAP, 0.6 mg l-1 NAA and 0.4 mg l-1 IAA. Biochemical analysis of healthy and diseased plants revealed that the contents of protein, phenol and chlorophyll were reduced in the inoculated plants as a result of phytoplasma infection. Carbohydrate content in brinjal increased immediately after inoculation and then decreased. The activity of peroxidase (PO) was enhanced in the inoculated plants while that of polyphenol oxidase (PPO) was reduced. The activity of phenyl alanineammonialyase (PAL) was reduced immediately after the inoculation, but enhanced at 30 and 60 days after inoculation (DAI). 91 92 The electrophoretic analysis of proteins using SDS-PAGE revealed the presence of two extra protein bands in the infected samples with molecular weights of 29 kDa (Kilo Dalton) and 43 kDa. The isozyme pattern analysis of peroxidase using native PAGE revealed two isoperoxidase bands in the inoculated plants with Relative mobility (Rm) values, 0.17 and 0.47, but a single band in healthy plants with Rm value of 0.17. Molecular detection was done using nested PCR. PCR products of ~1.8 kb (Kilo base) were obtained in direct PCR with phytoplasma universal primer pair P1/P7 and the nested PCR with P1/P7 followed by R16F2n/R16R2 amplified the fragment of size 1.2 kb. The presence of phytoplasma in tissue culture plants was also confirmed using nested PCR. Comparative nucleotide sequence analysis of brinjal and catharanthus isolates with the existing data base from NCBI revealed a 100% homology with brinjal little leaf phytoplasma isolates from Haryana and IARI and 99% homology with potato witches’ broom, potato purple top, tomato big bud phytoplasma etc. The 16S rDNA sequences of BLL and CLL phytoplasma shared 99.7% similarity with that of ‘Candidatus Phytoplasma trifolii (Ca. Phytoplasma trifolii)’. Thus the two phytoplasma isolates were identified as the related strains of ‘Ca. Phytoplasma trifolii’.
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, S
The 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 observed
Open Access
Immunological and molecular detection of banana viruses and production of disease free planting materials
(Department of Plant Pathology, College of Agriculture, Vellayani, 2014) Aliya, Ferzana; KAU; Umamahesaran, K
The study entitled "Immunological and molecular detection of banana viruses and production of disease free planting materials" was conducted in College of . Agriculture, Vellayani, and Thiruvananthapuram during !he period of2011-2014. Symptomatological studies showed that the characteristics symptoms caused by BBTV were small, brittle leaves with thickened veins which remained bunched at the top of the pseudostem. Plants with early infection did not produce fruits, where plants with later infection produce bunch with reduced size, weight and mishapen fingers. The characteristic symptoms caused by BBrMV were reddish spindle shaped lesion in the pseudostem, flag leaf sheath, leaf petiole, and bract. Leaves of infected plants showed characteristic chlorotic spindle shaped lesion on the leaf lamina. The characteristic symptoms of BSV were chlorotic streaks in the leaf lamina. Later the chlorotic streaks became necrotic. The characteristic symptom of CMV was mosaic pattern in the leaf lamina. The pathophysiological studies conducted in cultivar Nendran revealed that there was significant difference in carbohydrate, chlorophyll, protein and phenol content in infected plant when compared to healthy ones .. The activity of defence related enzymes like peroxidase, polyphenol oxidase and phenylalanine ammonialyase were found to be more in infected plants. Electrophoretic analysis of protein in virus infected samples through SDS-PAGE revealed the presence of an additional protein in the protein profile. The protein profile of BBTV infected sample showed one extra band with molecular weight of 20 kDa, BBrMV infected sample showed three additional protein band with molecular weight of 38 kDa, 29 kDa and 22 kDa, BSV infected sample showed three additional proteins with molecular weight of 25 kDa, 19 kDa, and 12 kDa, CMV infected sample showed one extra band with molecular weight of 25 kDa. Electrophoretic analysis of isozyme though native gel revealed the increased action of peroxidase enzyme in infected sample. Detection of VIruS infecting banana was carried out using varIOUS immunological techniques such as DAC-ELISA and DIBA using polyclonal antiserum (Agdia) and monoclonal antiserum. Both the techniques were found to be efficient in detecting virus infecting banana. Molecular diagnosis of the BBTV was carried out using CP gene and replicase gene specific primers. PCR product with amplicon size of about 530 bp was observed for coat protein gene specific primer where 237 bp was observed for replicase gene specific primer. Molecular diagnosis of BSV was carried out using two CP gene specific primers resulted in PCR product with amplicon size of 664 bp and 730 bp. Molecular diagnosis of CMV was canied out using CP gene specific primer resulted in PCR product with an amplicon size of 687 bp. CP gene specific primer for BBrMV did not give positive result. Cluster dendrogram analysis revealed that the BBTV isolate was mostly related to BBTV coat protein gene of Burundi isolate, BSV isolate was mostly related to banana streak virus isolate Trichi, CMV isolate was mostly related to cucumber mosaic virus isolate Trichi coat protein gene. The meristematic region of the virus infected banana suckers were excised and inoculated to MS media with BAP and NAA. The regeneration of plants from meristematic region was difficult because of high phenol production and contamination by endogenous bacteria. Meristem culture eliminated BBTV, CMV and BBrMV but not the BSV. Based on the research result, the banana VIruses can be detected usmg immunological and molecular technique and the meristem culture can eliminate all the banana viruses except BSV.

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