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Open Access
Integrated management of foliar fungal disease of culinary melon (Cucumis meloL. var. acidulus Naudin)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2016) Narmadhavathy, S; KAU; Kamala Nayar
The project entitled “Integrated management of foliar fungal disease of culinary melon (Cucumis melo L. var. acidulus Naudin)” was undertaken with the objective of making a comparative evaluation of the efficacy of foliar application of fertilizers, micronutrients, bio-control agents and newer fungicide for the management of Colletotrichum leaf spot (Colletotrichum sp.) disease of culinary melon. Surveys conducted during September 2013 to December 2013, in ten culinary melon fields located at Instructional Farm (IF), College of Agriculture (CoA), Vellayani as well as in farmers’ fields near, CoA, Vellayani, in order to assess the prevalence of major diseases such as Colletotrichum leaf spot and downy mildew disease affecting the crop. Highest disease incidence (DI) and percentage disease index (PDI) of Colletotrichum leaf spot were observed, 75 days after sowing, at Chavadinada (70.00 per cent and 64.44 per cent respectively). Incidence and index of downy mildew disease were recorded in four out of the ten locations surveyed (Palapoor, Papanchani, Kalliyoor and Punjakari). Maximum disease incidence and percentage disease index of downy mildew disease (36 per cent and 33.33 per cent respectively) were observed at Papanchani. The most virulent isolate of anthracnose leaf spot pathogen (IF, Vellayani isolate), obtained during the survey was identified as Colletotrichum fructicola by molecular characterization. The treatment NPK 19:19:19 (0.5 per cent) combined with the fungicide mancozeb (0.4 per cent) and adjuvant was most effective in inhibiting the mycelia growth of the pathogen C. fructicola, in vitro, (100 per cent) over control as well as in suppressing artificially induced anthracnose disease and improving the growth parameters of the plants, in the two greenhouse experiments conducted at the CoA, Vellayani during March to June 2014 and August to October, 2014. Results of two field trials conducted at CoA, Vellayani, during January to March, 2015 and April to June, 2015 for testing four most effective treatments screened from the greenhouse experiments, indicated that NPK 19:19:19 (0.5 per cent) + azoxystrobin (0.15 ml/l) + adjuvant (DI 40.00 and PDI 13.05 respectively) and NPK 19:19:19 (0.5 per cent) + mancozeb (0.4 per cent) + adjuvant (DI 40.00 and PDI 13.47 respectively) were most effective in managing the disease and also increasing total yield of plants, when compared to the remaining treatments. Trials were conducted in farmers’ fields at three locations (Venganoor, Vavamoola and Venjaramoodu) for confirming the efficacy of the two most effective treatments screened from the field trials conducted at CoA, Vellayani and pooled analysis of the results indicated that the lowest PDI (12.22) and DI (28.50) were obtained in plants treated with NPK 19:19:19 (0.5 per cent) + azoxystrobin (0.15ml/l) + adjuvant, which was significantly superior to the other treatments. Results of the microbial studies indicated that there was decline in fungal flora of the plants treated with foliar fertilizer NPK 19:19:19 (0.5 per cent) + azoxystrobin (0.15 ml/l) + adjuvant, days after application of treatments whereas bacterial population was higher in plants applied with the same treatment when compared to the application of combination of foliar fertilizer NPK 19:19:19 (0.5 per cent) + mancozeb (0.4 per cent) + adjuvant. There was indication of higher induction of systemic resistance in plants treated with NPK 19:19:19 (0.5 per cent) + azoxystrobin (0.15 ml/l) + adjuvant due to the higher activity of defense related enzymes, such as phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenol oxidase (PPO), β-1,3glucanase, super oxide dismutase (SOD) and the compound phenol, all of which, reached maximum level on the 15th day after treatment. Leaf samples obtained from plants treated with foliar fertilizer NPK 19:19:19 (0.5 per cent) + azoxystrobin (0.15 ml/l) + adjuvant indicated highest nutrient use efficiency in all three locations of the confirmation trials while highest pigment status due to this treatment was observed in the trial conducted at Venganoor. Relative water content was generally high in leaf samples collected from all plants irrespective of the treatments, although it was comparatively low, in leaf samples obtained from plants of absolute control plot. Epicuticular wax content was slightly lower in the plants treated with combination of the foliar fertilizer NPK 19:19:19 (0.5 per cent) and fungicides, either azoxystrobin (0.15 ml/l) or mancozeb (0.4 per cent) + adjuvant. Stomatal frequency on the upper and lower surfaces of leaves was not much affected by application of foliar fertilizer NPK 19:19:19 (0.5 per cent) combined with the fungicides. B:C estimated ratio revealed that the highest returns were obtained from the plants treated with foliar spray of NPK 19:19:19 (0.5 per cent) + azoxystrobin (0.15 ml/l) + adjuvant, in all three locations of the farmers’ field trials. This study presents the first report of the pathogen Colletotrichum fructicola causing anthracnose leaf spot disease of culinary melon in India. In field conditions, combination of the foliar fertilizer NPK 19:19:19 (0.5%) and azoxystrobin (0.15 ml/l) along with adjuvant applied twice at 15 days’ interval was most effective in controlling anthracnose leaf spot disease of culinary melon and also increasing the yield of the crop.
Open Access
Strain improvement of oyster mushrooms- pleurotus cystidiosus O.K. Mill and pleurotus opuntiae (Durieu and LEV.) SACC.
(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Krishnapriya, P J; KAU; Geetha, D
The present study entitled “Strain improvement of oyster mushrooms: Pleurotus cystidiosus O.K.Mill and Pleurotus opuntiae (Durieu and Lev.) Sacc.” was carried out in College of Agriculture, Vellayani during 2015-2018, with the objective to standardize the techniques for production of oyster mushrooms: P. cystidiosus and P. opuntiae; and to study their morphological, physiological and cultural characteristics as well as nutritional and organoleptic qualities; and to undertake genetic improvement by protoplast fusion. The mushrooms were collected from two locations of Thiruvananthapuram and three fast growing isolates of Pleurotus spp. viz., PC2 (Vellayani), PNC1 (Chirayinkeezhu) and PO1 (Vellayani) were selected for the study. These isolates were identified as P. cystidiosus subsp. abalonus, P. cystidiosus and P. opuntiae using internal transcribed spacer (ITS) primers and subsequent sequencing; and registered at Genbank database with accession numbers KY214254, KY887023 and KY214255 respectively. The fast growing isolates of P. cystidiosus (coremial), P. cystidiosus (non-coremial) and P. opuntiae recorded maximum growth on PDPA amended with one per cent yeast under dark condition. The optimum temperatures for the growth were 30 0C, 25 to 30 0C and 25 0C respectively whereas, the optimum pH were 8, 8 and 7 to 8 respectively. Studies with different substrates and amendments for spawn production revealed that sorghum with one per cent yeast was the best for P. cystidiosus (coremial) and P. opuntiae whereas, paddy grains with one per cent yeast for P. cystidiosus (non-coremial). Experiments with different substrates and amendments for mushroom production revealed that rubber wood sawdust sprayed with 2.5 per cent of 1 M potassium dihydrogen phosphate recorded the maximum BE for P. cystidiosus (non-coremial) (192.76 per cent). P. opuntiae recorded the maximum BE in rubber wood sawdust amended either with 4 per cent neem cake (91.38 per cent) or wheat bran (91.37 per cent). Major insect pests observed in the beds of Pleurotus spp. were phorid flies, spring tails, black ants and staphylinid beetles. The competitor moulds observed were different species of Coprinus, Aspergillus, Penicillium and Trichoderma. Sporocarps soaked in one per cent CA for 15 minutes followed by mechanical drying and powdering was the best post harvest treatment for both P. cystidiosus (non-coremial) and P. opuntiae. Mycelium of P. cystidiosus (coremial) showed black coremial structures, representing its asexual stage (Antromycopsis broussonetiae Pat. & Trab.). The coremia comprised of elliptical (16.31 µm x 7.48 µm) and round conidia (8.06 to 8.49 µm). The black colour of coremia was due to melanin which was extracted (255.56 mg l-1) and characterized. The performance of long duration P. cystidiosus (non-coremial) and short duration P. opuntiae was compared with two ruling mushrooms of Kerala viz., long duration P. florida (Mont.) Singer and short duration P. eous (Berk.) Sacc. The study revealed that P. cystidiosus (non-coremial) and P. opuntiae showed higher BE compared to P. florida and P. eous, respectively. P. cystidiosus (non-coremial) recorded maximum moisture (94.05 per cent), starch (200.55 mg g-1), protein (30.2 mg g-1), fat (4.25 per cent), antioxidants (485.45 μg equivalent gram of ascorbic acid-1), beta-carotene (25.69 µg 100 mg-1), polyphenols (7.55 mg g-1) and energy (359.45 Kcal) compared to other Pleurotus spp. Sensory evaluation of mushroom products made from the species of Pleurotus was done and masala curry prepared from P. cystidiosus (non-coremial) scored the maximum value for overall acceptability. Shelf life of P. cystidiosus (non-coremial) was higher (5 days) compared to P. opuntiae, P. florida and P. eous (3 days each) in perforated poly propylene covers stored under refrigeration. Vanillin (0.05 per cent) and carbendazim (1 mM) were selected as dual biochemical markers for the PEG mediated protoplast fusion. Three days old P. cystidiosus (non-coremial) and four days old P. opuntiae recorded the maximum protoplast yield at five and four hours after incubation respectively with 0.6 M KCl and 30 mg ml-1 of enzyme consortium. Eight fusant lines with varied mycelial characters were obtained. Among fusants, F6 and F8 did not segregate in the second generation whereas, F4 segregated. F6 and F8 recorded higher BE of 168.05 and 99.95 per cent respectively compared to the parental lines and other fusants. Sporocarp of F6 and F8 was morphologically similar to P. cystidiosus (non-coremial) and P. opuntiae respectively; and F8 also exhibited low temperature adaptability. The present investigation indicated the exploitability of two promising isolates viz. P. opuntiae for tropical areas and P. cystidiosus (non-coremial) for cooler regions of Kerala using locally available materials and the standardized cultivation practices. The present study also standardized the protoplast fusion technique between P. cystidiosus (non-coremial) and P. opuntiae; and two fusant lines (F6 and F8) recorded higher BE which can be used for future breeding programmes.
Open Access
Strain evaluation and production technology of shittake mushroom ( Lentinula edodes ( Berk. ) pegler)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2016) Deepa Rani, C V; KAU; Lulu Das
The present investigation on "Strain evaluation and production technology of Shiitake mushroom (Lentinula edodes (Berk.) Pegler' was conducted at Department of Plant Pathology, College of Agriculture, Vellayani, Thiruvananthapuram during the period 2012-2015. The aim of the experiment was to exploit various strains of Lentinula spp. for novel production technology and their phylogeny analysis through physiological and molecular studies. Surveys were collected during pre and post monsoon periods of May to December from different parts of Thiruvananthapuram, Kollam, Wayanad, Idukki, Pathanamthitta, Kannur and Kasargode districts. Six isolates of sp. (VLYN- 1 to VLYN-13) obtained during the survey were identified and compared with procured reference strains of Lentinula edodes (LE-1 to LE-5 from GB Pant University of Agricultural and Technology, Pantnagar, Uttarakhand) and LE-6 strain (Maharana Pratap University of Agriculture and Technology, Udaipur) . Morphologically the native isolates of Lentinus spp. had concave, funnel and convex pileus with varying colors and were leathery in nature.L. edodes strains in contrast had convex pileus with chocolate brown and golden yellow sporocarps which were fleshy and edible. Phylogenetic analysis of all six strains of L. edodes using RAPD markers confirmed the variability between the strains. Maximum similarity coefficient of 74.10 per cent was observed between LE-2 and LE-6 strains while LE-2 and LE-4 strains showed a minimum similarity coefficient of 35.70 per cent. Further studies by ITS sequencing showed that all the L. edodes strains tested in the study showed 99- 100 per cent similarity with the known sequences off L. edodes available in NCBI database while that of native isolates showed 99- 100 per cent similarity to Lentinus tuber-regium and Lentinus connatus thus confirming the variability between Lentinus and Lentinula sp. All the six strains of L. edodes, showed maximum mycelial growth in malt extract peptone dextrose agar in solid and oat meal broth in liquid medium. L. edodes strains preferred temperature of 20 °C with an acidic pH of 6. Dark and ambient light conditions favored maximum mycelial growth and biomass production for L. edodes culture. Although a minimum period of 16.33 days was required for full mycelial run in maize grains but due to comparatively less contamination rate in paddy grains which took 18.33 days for completion of mycelial run were selected as best substrate for further studies. Different substrates were evaluated for the development of a cultivation package for shiitake mushroom. Results showed that LE-1 strain took minimum of 71.00 days for initiation of sporocarp in sawdust supplemented with 20 per cent wheat bran. Hard wood sawdust especially of teakwood was used in the study. The substrate based on paddy straw and banana pseudo stem were not found effective for pinhead initiation and thus failed to produce sporocarps. LE-1 produced maximum sporocarp (11.33) in sawdust + 20 per cent wheat bran which was followed by LE-3 (10.63) in sawdust + 20 per cent rice bran. Maximum yield of 290.66 g/ 500 g substrate was obtained in sawdust + 20 per cent wheat bran by LE-6 strain. Maximum biological efficiency of 58.13 per cent was also recorded in LE-6 in sawdust supplemented with 20 per cent wheat bran substrate. Substrates like paddy straw and sawdust amended with 20 per cent wheat bran substrates were evaluated for the development of native isolates of Lentinus tuberregium and Lentinus connatus . Results showed that maximum biological efficiency of 58.00 per cent was obtained by Lentinus tuber-regium whereas 36.60per cent biological efficiency by Lentinus connatus in sawdust amended with 20 per cent wheat bran substrate. Nutrient analysis of all the six strains showed that carbohydrate content ranged between 35.29 per cent to 40.23 per cent, protein 18.33 per cent to 21.66 per cent, crude fibre 22.33 per cent to 27.33 per cent, Vitamin- C 2.53 per cent to 3.50 per cent, ash 2.70 per cent to 4.40 per cent and lipid 2.46 per cent to 3.60 per cent. Mineral content of L. edodes included Ca (11.00 mg to 19.00 mg/ 100 g), Mg (0.46 to 1.10 mg/ 100 g), Fe (1.36 mg to 1.80 mg/ 100 g), Mn (1.53 mg to 2.63 mg), P (1.65 mg to 2.87 mg), K (16.33 mg to 25.20 mg), Na (13.00 mg to 23.66 mg) and Zn (19.66 mg to 28.33 mg/ 100 g). Sensory evaluation of mushroom products made from L. edodes was carried out by a panel of judges for various characters of which mushroom masala scored maximum for texture, taste, flavor and overall acceptability when compared to other recipes like mushroom cutlet, scramble, soup, baji and biscuit. As part of the study, paddy grain was found to be the most suitable substrate for spawn production of L. edodes and teakwood sawdust amended with 20 per cent wheat bran was the most efficient bed substrate. LE-6 strain was superior in terms of yield and biological efficiency. Therefore findings of the above investigation recommends the adoption of a suitable cultivation package for shiitake mushroom by using low cost substrates (hardwood sawdust) available in Kerala in plains and hilly regions.
Open Access
Endophytic microorganism mediated systemic resistance in Cocoa against Phytophthora palmivora (Butler) Butler
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2011) Sainamole Kurian, P; KAU; Koshy Abraham
The study on 'Endophytic microorganism mediated systemic resistance in cocoa against Phytophthora palmivora (Butler) Butler was carried out during 2005-2010. The pathogen causing pod rot of cocoa was isolated from infected pods , and its pathogenicity established. Based on cultural and morphological characters, it was identified as Phytophthora palmivora (Butler) Butler. Endophytes were isolated from samples of feeder roots, tender shoots, leaves and pods of cocoa collected from various locations of major cocoa growing area of the state. The population of endophytic microflora varied among different locations and parts of the plant, and in general, the population was more in roots. Bacteriaand fluroscent pseudomonads were more abundant than filamentous fungi and yeasts. Out of the 325 endophytic isolates comprising of 116 bacteria, 153 fluorescent pseudomonads, 34 years and 22 fungi, 82 were found exerting antogonism towards the pathogen. These antagonistic endophytes were further evaluated in In vitro by dual culture and by inoculation on detached cocoa pods, and leaves. It was found that, 25 isolates were more efficient antagonists.
Open Access
Integrated management of viral diseases of bittergourd (momordica charantia L.)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2018) Radhika, N S; KAU; Umamaheswaran, K
The present research work entitled ‘Integrated management of viral diseases of bitter gourd (Momordica charantia L.) was carried out in the College of Agriculture, Vellayani during 2014-2017, with the objectives to study the occurrence and distribution of viruses in bitter gourd in Thiruvananthapuram, Idukki and Palakkad, immunomolecular characterization of the viruses, and screening of antiviral chemicals, antiviral principles of animal, plant and microbial origin for the management of the disease. In the suvey conducted at five locations in Thiruvanaanthapuram district, Pappanchani area recorded highest incidence of viral disease (60%) while highest Vulnerability Index (V.I) was recorded from Vellayani (56.00). In Idukki district, six major bitter gourd cultivating areas were surveyed among which Rajakumary area recorded the highest disease incidence (100%) and V.I (82.00). In Palakkad district, five locations were surveyed, among which panackatri and Thekkepotta recorded highest disease incidence of 88% and highest V.I (69.00). The major insects associated with the crop were whitefly (Bemisia tabaci (Genadius) with an incidence of 10-25%, aphids (Aphis gossypii glover) with an incidence of 10-40%, Jassids (Empoasca (Empoasca) motti Pruthi) with an incidence of 10-30% and mites with an incidence of 10-50%. Phyllody and little leaf symtoms (20% incidence) were also recorded in bittetgourd form Rajakumary and Rajakkad areas in Idukki. Flat limb and multiple proliferation of shoot tip were observed at many fields in Idukki. Symptoms associated with the disease include yellow mottle, mosaic,blistering, leaf curl and reduction in leaf size. Yellow mosaic and blistering is seen in severe infection finally leading to stunting of the plant, reduced flowering an fruiting and hairyness on stem. Mechanical transmission of the virus on Datura stramonium produced yellow lacal lesions indicating the presenceof Bean Golden mosaic virus (Begomo) in the infected leaf extract. This leaf extract also produced local lesions on othe indicator hosts like Chenopodium amaranticolor and Gomphrena globosa indicating the presence of Cucumber mosaic virus (CMV) or Potato virus Y (PVY). The viruses were transmitted by whiteflies (20%) and aphids (30%) from infected bittetgourd plants to healthy seedlings. Whiteflies (Bemisia tabaci Gennadius)) and aphids (Aphis gossypii Glover) are the vectors of the respective viruses Wedge grafting diseases scion on to 3-5 leaf stage healthy seedling of bittergourd produced symptoms of infection within ten days. KAU varieties Preethi and Priyanka were found to be susceptible to infection with preethi expressing a V.I of 70.80 and Priyanka expressing a V.I of 62.50 respectively. Ensyme linked immunosorbent assay (ELISA) and Dot immunobinding assay (DIBA) revealed the presence of three viruses belonging to Begomo, CMV and PVY group causing an mixed infection in bittergourd. The presence of all the three viruses were also confirmed in electron micrograph, Begomovirus as twin particles of size 18-20 X 30nm,CMVas single particles of 18nm and PVY as lonog flexuous rod of size 750nm. PCR amplification of coat protein gene (cp gene) of virus isolates from all the three districts yielded an amplicon of size approximately equal to 570 bp. Idukki and Palakkad isolates showed 94% identity to Tomato leaf Curl Virus isolate TNUDU BGI Coat Protein (AVI) gene while Trivandrum isolate showed 95% identity to Tomato leaf Curl Virus isolate TNPDU BG4 Coat Protein (AV1) gene . Phylogenetic tree constructed using multiple sequence alignment programme showed close relation between Begomo viruses identified in bittergourd from different districts. Studies on defense related enzymes such as peroxidase (PO), polyphenol oxidase (PPO) and phenyl alanine ammonialyase PAL) showed significant activity of PO and PPO in diseased plants than in healthy plants and the activity was on par in healthy and diseased for PAL. Protien profile of healthy and diseased at different days after virus inoculation through grafting indicated the production of novel proteins in diseased. There was no difference in the native profile of peroxidase in healthy and diseased at 15 days after virus inoculation. An additional isozyme band with a Rm value of 0.5 was observed in diseased at 45 days after virus inoculation. Management of the disease with antiviral chemicals and antiviral principles of plant, animal and microbial origin was undertaken as pot culture studies with pre and post inoculation of treatments. Twelve treatments with three replications each were laid out in completely randomized design for the evaluation. The treatments included Aspirin at two levels of 100 and 150 ppm, Salicylic acid (SA) at two levels of 100 and 150 ppm and Acibenzolar S methyl (ASM) at 50 and 75 ppm concentration, and two commercial formulations viz., Perfect and virus –Ex at 0.5 and 1.0 ml concentrations. The treatments were applied three times at 10 days interval. Pre application of thrice sprapying of Acibenzolar S methyl (ASM), 75 ppm concentration (V.I-35.00) at ten days interval was statistically significant over other treatments followed by ASM-50 ppm (V.I-41.33). Post application of antiviral chemicals also showed a statistically significant effect of three times spraying ASM-50 ppm(V.I-25.00) at ten days interval followed by spraying of Virus Ex 1ml L-1 (VThe best eight treatments with control was laid out as Randomised Block Design at the Instructional Farm, College of Agriculture, Vellayani during February to May 2017 as a field trial to study the effect of treatments on natural incidence of the viruses in the susceptible variety Preethi. The treatment, three sprays of ASM-50 ppm (V.I-28.33) at ten days interval ws on par with buttermilk (Three times dilution of curd) (V.I-39.16). Yield was also significantly high in ASM-50 ppm (437g plant-1) followed by Pseudomonas fluorescens talc based formulation (2%) (233 g plant-1)among the treatments.
Open Access
Identification of graft transmissible resistant factors and development of si RNA mediated resistance in cassava against cassava mosaic geminivirus
(Department of Plant Pathology, College of Agriculture, Vellayani, 2017) Asha, B Nair; KAU; Umamaheswaran, K
The present study entitled ‘Identification of graft transmissible resistant factors and development of siRNA mediated resistance in Cassava against Cassava mosaic virus’ was carried out during the period 2012-2017 at the Department of Plant Pathology, College of Agriculture, Vellayani. The study was carried out with the objective of identification of transferability of resistance factor from resistant cassava, Sree Padmanabha to susceptible, Vellayani Hraswa by grafting and to develop siRNA mediated technology for the development of cassava plant resistant to Cassava mosaic geminivirus. Grafting experiments were conducted using resistant Sree Padmanabha as root stock and susceptible Vellayani Hraswa as scion. Symptoms like leaf mosaic, chlorotic spots, reduction in leaflet size and stunting of plants were noticed in susceptible variety. Virus concentration was found to be less in grafted plants. Grafting experiments showed the expression of an extra protein by SDS-PAGE and Coomassie staining in grafted plants which is around 38 kDa. Molecular weight of the new protein revealed the presence of extracellular protein in grafted samples. The extra proteins found in the grafted plants are assumed to be transferred from Sree Padmanabha to Vellayani Hraswa by the process of grafting. The study also involved the development of an intron hairpin RNA vector against replicase gene of SriLankan cassava mosaic virus and introduction of this construct into embryogenic cells via Agrobacterium mediated transformation. A protocol for somatic embryogenesis in cassava variety, Vellayani Hraswa was developed by using immature leaf lobes as explants. The young leaf lobes from tissue culture plantlets produced through meristem culture was used for embryogenic callus formation. Cremish white calli was initiated in Murashige and Skooge (MS) medium supplemented with picloram 12 mg L-1 in dark. For embryogenesis, the calli were transferred to MS medium supplemented with BA 2µM and NAA 1µM which resulted in the production of glassy elongated somatic embryos. The germinated cotyledonary embryos were then regenerated into plantlets by culturing in MS medium supplemented with BA 1mg L-1. Effort was taken to construct an intron hairpin RNA vector and the gene targeted for silencing was the replicase gene of SriLankan cassava mosaic virus (SLCMV). Total DNA was extracted from virus infected plants and the whole replicase gene was isolated using gene specific primers. Sequencing of the whole gene was done. BLAST analysis showed 98% similarity to replicase gene of various isolates of SLCMV. The sequence was then subjected to miRNA target prediction and restriction mapping to select suitable region for the construct. Based on this information, a fragment of 397 bp towards the 5’ end was amplified by designing a set of primers with anchored restriction sites. The primers anchored with Xho and Kpn 1sites were used for the amplification of sense strand and the primers anchored with Xba and Cla 1sites were used for amplification of antisense strand. Selected region was amplified to form sense and anti-sense fragments and cloned to pTZ57R/T cloning vector. Inserts were then released from pTZ57R/T using the corresponding restriction enzymes. The sense and anti-sense fragments were then integrated in the primary vector pHANNIBAL on either side of the pdk intron which facilitated the formation of intron hairpin RNA construct. The intron hairpin RNA construct in pHANNIBAL contained CaMV35S promoter, sense strand, pdk intron, antisense strand and OCS terminator in the order with Not 1 restriction sites. After confirmation of integration by restriction digestion, the Not1 fragment with sense and anti-sense strand were released from pHANNIBAL and ligated to the digested Not1 site in the lacZ gene of binary vector pART27 containing antibiotic resistant marker nptII and spec. the binary vector was confirmed for the presence of insert by transferring to DH5α cells and colony selection by blue white screening. Plasmid DNA isolated from transformed colonies grown on Luria agar medium supplemented with 100 mg L -1 spectinomycin were confirmed for the presence of insert. After confirmation of insert in the binary vector, it was transformed to Agrobacterium tumefaciens strain LBA4404 via freeze thaw method. Transformed colonies were selected on kanamycin selection medium at 100 mg L -1 and confirmed for the presence of binary vector and ihpRNA insert using nptII primers and primer for sense and antisense strands by PCR reaction. Cotyledons excised from the somatic embryos were transformed with LBA4404 having pART 27 by co-cultivation and the transformed embryos were selected with antibiotic pressure (Kanamycin 100 mg L-1). DNA was isolated from the transformed somatic embryos and confirmed for the presence of insert using forward primer of sense fragment and reverse primer of antisense fragments. Transformed embryos were subjected to regeneration.
Open Access
Molecular characterization of virus causing infectious chlorosis disease of banana
(Department of Plant Pathology, College of Horticulture Vellanikkara, 2017) Ahamed Mujtaba, V; KAU; Anita Cherian, K
The experiment entitled “Nutrient management in strawberry (Fragaria x ananassa Duch.)” was undertaken at Regional Agricultural Research Station, Ambalavayal, Wayanad during the year 2016-17. Performance of strawberry variety Winter Dawn was evaluated under nine treatments and a control in the open field viz., FYM 10 t ha-1 + NPK 50:20:50 kg ha-1 (T1); FYM 10 t ha-1 + NPK 75:30:75 kg ha-1 (T2 ); FYM 10 t ha-1 + NPK 100:40:100 kg ha-1 (T3); FYM 20 t ha-1 + NPK 50:30:100 kg ha-1 (T4); FYM 20 t ha-1 + NPK 75:40:50 kg ha-1 (T5); FYM 20 t ha-1 + NPK 100:20:75 kg ha-1 (T6); FYM 30 t ha-1 + NPK 50:40:75 kg ha-1 (T7); FYM 30 t ha-1 + NPK 75:20:100 kg ha-1 (T8); FYM 30 t ha-1 + NPK 100:30:50 kg ha-1 (T9) and an absolute control (T10), without any nutrient application. All the treatments were on par and superior over the control (T10) in case of plant height. In case of plant spread, T2, T3, T5, T6, T7, T8 and T9 were on par and superior over the control while T1 and T4 were on par with each other but differs with other treatments. All the treatments except T2 were on par and superior over the control with respect to number of leaves per plant. Application of treatments had no significant effect on days to first flowering. In case of number of flowers and clusters per plant, T1, T2, T3, T5, T6, T7, T8 and T9 were on par and superior over the control while T4 was on par with the control (T10). Days to first harvest was minimum in T6, T7, T8 and T9 which were on par while all other treatments were on par with the control (T10).In case of number of fruits and yield per plant, T7 (FYM 30 t ha-1 + NPK 50:40:75 kg ha-1) and T8 (FYM 30 t ha-1 + NPK 75:20:100 kg ha-1) were on par and superior over other treatments including T1, T2, T3, T4, T5, T6 and T9 which were on par and superior over the control. Average fruit weight recorded under T3, T5, T6, T7, T8 and T9 were on par which was followed by T2 on par with T4 and T1. Days to final harvest was not found to be influenced by the application of different treatments. Biochemical characters of fruits viz., TSS, acidity and TSS/acidity ratio were not having any significant effect due to the application of treatments. In case of total sugars, T3, T7, T8 and T9 were having the highest content and were on par which was followed by T5 on par with T1, T2, T4, T6 and T10. The overall sensory score was highest in T7 followed by T8. Application of different treatments had no significant effect on the shelf life of strawberry fruits. N, P, K and Ca content in the plant were not significantly affected by any treatment while Mg content was found to be on par in all treatments and superior over the control. Soil analysis after the harvest of the crop revealed that the values for soil EC, available P, K, Mg and S were found to be elevated while soil pH, organic carbon and available Ca content were found to be at lower levels than the initial values before planting. It was concluded that among different nutrient combinations evaluated, T7 (FYM 30 t ha-1 + NPK 50:40:75 kg ha-1) with a BC ratio of 3.06 can be recommended for further optimization and refinement.
Open Access
Enhancement of systemic resistance to soil borne pathogens of ginger by enriched spent mushroom substrate of pleurotus sajor-caju
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2017) Remya, J S; KAU; Beena, S
Spent mushroom substrate (SMS) is the composted organic material retained after a crop of mushroom. The world mushroom industry needs to discard more than 50 million tons of SMS every year. The latest research throw light on the efficient use of SMS for the disease management of crop plants. A preliminary study on the use of SMS of Pleurotus spp. as mulch for the management of rhizome rot complex disease of ginger under pot culture condition was carried out in the Department of Plant Pathology, College of Horticulture, Vellanikkara. In this study, among the various SMS used, the paddy straw SMS of P. sajor-caju as mulch recorded the highest biometric characters and least disease incidence compared to control. Hence this project was proposed as the continuation of the above study to evaluate the efficacy of enriched SMS of P. sajor-caju in enhancing growth and systemic resistance for the management of soil borne pathogens of ginger under field conditions. SMS of P. sajor-caju was produced during three different periods viz., March-April, June-July and November-December (2013) at six different locations of Kerala viz., College of Horticulture, Vellanikkara (Thrissur dist.), farmer’s field at Kodakara (Thrissur dist.), Perinjanam (Thrissur dist.), Krishnagiri (Wayanad dist.), Mananthavady (Wayanad dist.) and College of Agriculture, Vellayani (Thiruvananthapuram dist.). Enumeration of microflora of these SMS was carried out and a total of 47 fungal and 45 bacterial isolates were obtained. The antagonistic efficiency of these isolates were evaluated against the five pathogens viz., Pythium aphanidermatum, Fusarium oxysporum, Rhizoctonia solani, Sclerotium rolfsii and Ralstonia solanacearum under in vitro conditions. All the isolates from SMS showed antagonistic property against one or the other soil borne pathogens, with varying degree of inhibition. Mutual compatibility between the most efficient fungal and bacterial antagonists was evaluated to develop an effective microbial consortium for enriching the SMS and could be used against the soil borne diseases of ginger. Biosoftening efficiency of selected fungal and bacterial antagonists on SMS was evaluated. Two separate experiments were carried out with the selected antagonists effective against fungal and bacterial pathogens. For each experiment, five fungal antagonists, five bacterial antagonists and three compatible pairs were selected based on the in vitro evaluation of antagonistic efficiency and mutual compatibility studies. SMS was enriched separately with different antagonists and standardized the period for biosoftening of SMS as mulch for ginger cultivation. A period of 15 days was selected as most suitable for biosoftening the SMS as mulch with optimum antagonistic fungal and bacterial population. Wide range of C:N ratio was recorded by the SMS enriched with each antagonists. By considering the C:N ratio along with external appearance, the treatments having C:N ratio of 30:1 to 45:1 were selected, since it was the most suitable stage to be used as mulch in ginger. The effectiveness of biosoftened SMS against rhizome rot and bacterial wilt diseases of ginger was evaluated in two pot culture experiments. Three fungal and bacterial antagonists each and one compatible pair of antagonists which were selected based on the in vitro evaluation of antagonistic and biosoftening property were used for enriching the SMS. After enrichment, the SMS were kept for 15 days for biosoftening and were applied as mulch in the experiments. Observations on germination percentage and other growth parameters viz., number of tillers/plant, number of leaves/tiller and height of tillers were recorded at one month intervals from two months after planting (MAP). Challenge inoculation of pathogens was done at 45 days after germination and per cent disease incidence was recorded at 7 and 14 days after inoculation (DAI). In the experiment for the management of P. aphanidermatum, the lowest disease incidence was observed in T7 (SMS softened with P1F1+ M1F2) on 14th day after inoculation (DAI). This treatment also recorded the highest number of tillers, number of leaves/ tiller and height of tillers and rhizome yield. In the experiment for the management of R. solanacearum, the treatment T2 (SMS softened with T1F2) was found to be the most efficient one, which recorded the least disease incidence at 14 DAI, whereas the highest values for biometric characters and rhizome yield were recorded by T7 (SMS softened with K1B1 + T2B1). The activity of phenol and defense related enzymes such as peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) were estimated by spectroscopy, before challenge inoculation with pathogen and at one, three and five days after the challenge inoculation. For estimation, leaf samples were collected separately from the pot culture experiments I and II for the management of P. aphanidermatum and R. solanacearum respectively. The treatments which recorded less disease incidence in both the pot culture experiments exhibited the highest activity of defense related enzymes and phenol. The highest activity of defense related enzymes and phenol was recorded at 5 DAI. Thus the present study showed that in addition to direct antagonism and plant growth promotion, induction of defense related enzymes was also contributed by SMS to enhance resistance against invasion of soil borne pathogens of ginger. Field evaluation of the selected treatments from pot culture experiments showed the lowest disease incidence in the treatment T2 (SMS softened with T1F2) followed by T5 (SMS softened with P1F1+M1F2). Statistically these were on par with each other. The treatment T5 (SMS softened with P1F1+M1F2) recorded the highest germination percentage, number of tillers, number of leaves/tiller and rhizome yield also. Analysis of primary nutrients viz., nitrogen, phosphorus and potassium in SMS, plant and soil from field experiment was conducted. Among these, SMS softened with P1F1+M1F2 recorded the highest percentage of N, P and K. This treatment recorded the highest nutrient content in ginger rhizome and soil also. Attempts were also made to identify the fungal and bacterial antagonists selected for field experiment. Based on the cultural and morphological characters, the fungal antagonists viz., Kr1F4, T1F2, P1F1 and M1F2 were tentatively identified as Trichoderma viride (Pers.), T. viride (Pers.), T. koningii (Oudem.) and T. harzianum (Rifai) respectively. The identification got confirmed from National Centre for Fungal Taxonomy (NCFT), New Delhi. The bacterial antagonists selected for field experiment were also identified based on cultural, morphological, biochemical and 16s rRNA sequence analysis. The three bacterial antagonists P3B2, T2B1 and K1B1 were identified as Bacillus safensis, B. methylotrophicus and Burkholderia gladioli respectively. Spent mushroom substrate is rich in microflora and these microflora exert antagonistic activities against soil borne pathogens. It stimulates the natural defense system in plants, provide necessary nutrients for plant growth and also improve soil physical condition. From field evaluation it was found that the SMS softened with T. viride recorded the lowest disease incidence and which enhanced systemic resistance to soil borne pathogens of ginger by defense related enzymes and phenol. The results were on par with the SMS softened with consortium of antagonists, T. koningii and T. harzianum (P1F1+M1F2). The highest rhizome yield and other growth parameters were also contributed by the SMS softened with T. koningii and T. harzianum. The content of nitrogen, phosphorus and potassium were also recorded the highest in this SMS. So from the present study it can be concluded that the SMS softened with T. koningii and T. harzianum can be used as mulch in ginger which was found equally effective to induce systemic resistance against soil borne pathogens and to enhance growth parameters and rhizome yield.
Open Access
Characterization and exploitation of jelly mushrooms (auricularia spp./ Tremella spp.)
(Department of Plant Pathology, College of Agriculture, Vellayani, 2017) Priya, R U; KAU; Geetha, D
The present study entitled “Characterization and exploitation of jelly mushrooms (Auricularia spp./ Tremella spp.)” was carried out in College of Agriculture, Vellayani during 2014-2017, with the objective of standardization of techniques for production of jelly mushrooms (Auricularia spp./ Tremella spp.) in agricultural wastes and to study their morphological, physiological and cultural characteristics as well as nutritional and organoleptic qualities. Survey was conducted in ten different locations of Thiruvananthapuram and Kollam districts of Kerala during 2014-2016. Sporocarps of Auricularia spp. and Tremella spp. were collected from tree stumps, predominantly Mango, Coconut, Drumstick, Teak wood and Rubber. All the mushrooms collected from all the locations were gregarious in nature and lignicolous in habitat. Morphological studies of jelly mushrooms showed that the sporocarps were light brown to dark brown in colour with incurved margin, ear shaped, soft and velvety in texture and devoid of stipe. The internal stratification of hyphae showed eight different zonations. Basidiospores were hyaline, oval and sub cylindrical to cylindrical shaped. Two fast growing isolates selected based on the time taken for complete mycelial growth and nature of mycelial growth, designated as A1 and A2 were sent to Directorate of Mushroom Research, Solan for identification. These were identified as Auricularia polytricha (Mont.) Sacc. (Accession number of A1 was DMRO-825 and A2, DMRO-826). The maximum mycelial growth was recorded on malt extract agar medium and a temperature of 250C, pH 7 and light conditions were found favourable for mycelial growth. Evaluation of different substrates for spawn production revealed that paddy grain was the best substrate followed by sorghum. Rubber sawdust spawn recorded maximum keeping quality. Malt extract broth was found to be the best for submerged culture production of both A1 and A2. Evaluation of different substrates for mushroom production revealed that rubber sawdust was the best substrate for cultivation which recorded maximum Biological Efficiency (BE) of 14.8% for A1 and 12.2% for A2. The minimum time for spawn run was taken by paddy straw and the maximum was taken by neopeat. Major insect pests observed were Megaselia sp., Seira sp. and Staphylinus sp. The competitor moulds observed were Coprinus sp, Aspergillus spp., Penicillium sp. and Trichoderma sp. Among the different amendments, wheat bran (2.0 and 4.0 %) and groundnut cake (2.0%) were found to be the best for enhancing the growth parameters whereas, rice bran (2.0% and 4.0%) was the best for increasing yield parameters. Analysis for the proximate constituents in A. polytricha (A1 and A2) revealed that it contained appreciable amount of carbohydrate (47.1 and 48.8%), protein (18.06 and 20.75%), polyphenols (9.53 and μg), fibre (17.69 and 15.49%), total anti oxidants (116 and 74μg), β carotene (0.178 and 0.150 μg) and the energy value was 251.49 and 264 respectively. Sensory evaluation of mushroom products made from A. polytricha (A1 and A2) indicated that mushroom tomato sauce scored maximum for overall acceptability. Under refrigeration (40C) in perforated poly propylene covers mushrooms could be kept fresh for three days. Indoor cultivation of both A1 and A2 showed significant results for growth parameters as well as yield parameters compared to outdoor cultivation. Comparative performance of A. polytricha with two ruling mushrooms of Kerala namely oyster mushroom (Pleurotus florida (Mont.) Singer.) and milky mushroom (Calocybe gambosa (Fr.) Donk.) indicated that oyster mushroom took minimum days for spawn run, pinhead formation and first flush compared to milky mushroom and A. polytricha. Milky mushroom recorded highest BE (66.1%) compared to others. Studies on medicinal activities of A. polytricha indicated that it possessed anticancerous activities for cervical, colon and liver cancer cell lines and higher activity was recorded in 100 μg/ml concentration. Based on the results of present investigation, A. polytricha can be cultivated successfully in tropical areas on locally available materials. Paddy grain was the most suitable substrate for spawn production and Rubber sawdust amended with 2% rice bran, the most suitable growing medium. Mushrooms possessed significant nutritional and medicinal activities. The findings of the above investigations recommend the adoption of a suitable cultivation package for jelly mushrooms (A. polytricha), a highly prized mushroom.

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