Browsing by Author "Sanju Balan"

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Characterisation and management of sugarcane bacilliform virus (SCBV) causing leaf fleck disease in sugarcane
(Department of Plant Pathology, College of Agriculture Vellanikkara, 2021) Sanju Balan; Anita Cherian
Sugarcane (Saccharum officinarum) is a monocotyledonous perennial cash crop cultivated worldwide both under tropical and sub tropical conditions. It is being cultivated in more than 120 countries in the world. Like any other crops, it is also susceptible to biotic stress. Of which, diseases caused by viruses not only pose serious threat to sugarcane cultivation but also result in deterioration and exclusion of elite varieties of the germplasm. One of the major viral disease which affects global exchange of sugarcane germplasm is leaf fleck disease caused by Sugarcane bacilliform virus (SCBV). The research project entitled ‘Characterization and management of Sugarcane bacilliform virus causing leaf fleck in sugarcane’ was initiated with purposive sampling surveys in selected sugarcane fields in districts of Kerala and Tamil Nadu in order to document the symptoms under natural conditions, to assess the disease incidence, severity and to collect infected samples for further studies. The per cent disease incidence of the leaf fleck disease in Kerala ranged from 12 to 51 per cent whereas severity ranged from 10 to 36.5%. In Tamil Nadu the per cent disease incidence ranged from 28 to 56 per cent while severity ranged from 28 to 50.41%. Major symptoms observed on leaves were mottling, chlorotic flecks, chlorotic patches streaks and stripes with general yellowing of the canopy. In the case of severely affected clones, there was reduction in tillering, internodal length, number of internodes and appearance of deep longitudinal cracks. In highly susceptible clones, stunted growth with bunchy top appearance was noticed. On the basis of phenotypic variability of symptom expression, genotypes were classified into five groups. The development of the symptoms was also studied under artificial condition through insect transmission of the virus using pink mealy bug, Saccharicoccussacchari. Morphological characterisation of the virus done using electron microscopy revealed the presence of bacilliform virus particles of size 30 X 130–150 nm which indicated that the virus belongs to genus BADNA and family Caulimoviridae and the etiology of the disease was confirmed as Sugarcane bacilliform virus. The molecular detection of SCBV was also standardized through polymerase chain reaction (PCR). PCR amplification of RNaseH/RT gene was done using BADNA specific and SCBV129 specific primers. The amplicons were sequenced and in silco analysis of sequences showed sequence homology of 99 to 100 percent identity to SCBV. Widespread occurrence of the disease was observed even in the early generation of varietal development and in newly developed varieties. The transmission of the virus was suspected through true seed (fluff) developed by biparental crossing during sugarcane varietal development programme. Hence, the study was conducted to establish possible transmission of the virus from sugarcane parents to their progenies and the role of maternal and paternal parents in disease transmission through true seeds to the progenies. Samples from eight months old seedlings, three months old seedlings and parental clones were tested positive to the virus in PCR assays. Real time PCR was also standardized to assay these clones. Immunodiagnostic technique was validated using DAC ELISA. The technique of immunocapture PCR was also standardized. Minimal dilution of antisera with which SCBV could be detected was 2:1000 (V/V). Plant extract (antigen) at a dilution of 1:5 was found to be optimal for the detection of SCBV. Molecular detection of SCBV from mealy bug vector was also standardized. Both phenotypic and molecular methods were utilized to identify potential sources of natural resistance against SCBV. Based on the severity of symptom expression and PCR assays these were further classified as highly susceptible (HS), moderately susceptible (MS) moderately resistant (MR) and resistant (R). For generation of RNAi hair pin construct, initially forward (SF) and reverse primer (SR) were used to amplify 700 bp fragment of RT/RNase H gene to be cloned in sense orientation of the vector, pHANNIBAL. The linearized vector and the insert were ligated, and the ligation mixture was used to transform competent cells of Escherichia coli and the transformants were selected. Later antisense forward (AF) and reverse (AR) primer pairswere used to amplify 700 bp fragment of RT/RNase H gene to be cloned in antisense orientation. PCR product ligated into antisense direction of the vector and transformed into competent cells of E. coli. The recombinant pHANNIBAL vector was digested with restriction enzymes. The recombinant pHANNIBAL vector harbouringRNase H /RT gene was released from the vector through Not I site and subcloned into plant expression binary vector. Thus, cassette for RNA silencing was prepared.130 Meristem tip culture was also standardized with antiviral chemical tenofovir. Recovery percentage of meristem varied from 70 to 75 per cent and the viral load was quantified using real time PCR. The outcome of the study would facilitate early detection and elimination of the source of infection and prevent the spread of the disease in the field. Information generated in the study could be utilized while planning biparental crossing and reduce the spread of the virus in varietal development programmes. The hair pin construct developed in this study could be further utilized to generate transgenic disease resistant plants.
Potential of antagonistic endophytes against bacterial blight of anthurium
(Department of Plant Pathology, College of Horticulture, Vellanikkara, 2009) Sanju Balan; Koshy Abraham
One of the major constraints in anthurium cultivation is the severe incidence of bacterial blight disease. The pathogen causing bacterial blight of anthurium was isolated and identified as Xanthomonas axonopodis pv. dieffenbachiae based on its cultural, morphological and biochemical characters coupled with its pathogenicity. Endophytic microbes were isolated from different parts of healthy anthurium collected from various locations of Thrissur, Kannur, Kasargod and Thiruvananthapuram districts. Isolation yielded more number of bacteria than fungi. Out of 51 endophytes tested, only eight bacterial and two fungal isolates showed antagonism against the pathogen. The eight selected bacterial endophytes were subjected to various tests for understanding the parameters that may act to produce antagonism as well as enhanced growth of the plants. The antagonists varied in their ability to promote plant vigour, hydrogen cyanide, IAA, ammonia and siderophore production and Phosphorus solubilization capacity. The endophytes were compatible with Bavistin, Akomin and Contaf and incompatible with Indofil M 45, Saaf and Captaf. Six insecticides viz., Classic, Rogor, Ekalux, Malathion, Target and Hostathion, and four fertilizers viz. Muriate of potash, Rajphos, Urea and Factomphos were compatible with the isolates. However, the isolates exhibited variation in their sensitivity with the antibiotics tested. Further, all isolates were compatible to each other. A pot culture experiment was conducted to assess the field efficacy of selected endophytes in comparison with recommended management practices. The treatments were given as two pre inoculation and two post inoculation sprays. The incidence and severity of the disease were recorded at five days interval for a period of 30 days. The result indicated that plants treated with endophyte EB15 showed minimum disease incidence and severity. This was followed by treatment with EB 31 and Streptocycline. The promising endophytes, EB15 and EB 31 were tentatively identified as Bacillus sp. and Pseudomonas sp. respectively.