G. Karthikeyan

Induction of systemic resistance in blackgram (Vigna mungo) against urdbean leaf crinkle virus by chemicals

Abstract The effect of chemicals on the induction of systemic resistance in blackgram (Vigna mungo) against urdbean leaf crinkle virus (ULCV) was studied. Pre-inoculation spraying of blackgram plants with resistance-inducing chemicals, namely salicylic acid and benzothiadiazole at 100 ppm concentration was found to be effective in reducing ULCV infection and increasing the incubation period of the virus under controlled conditions. The treatment of blackgram plants with salicylic acid and benzothiadiazole significantly increased the phenylalanine ammonialyase (PAL), peroxidase (PO) and polyphenol oxidase (PPO) activities. New isoforms of peroxidase and polyphenol oxidase were also induced by these treatments. A two-fold increase in accumulation of total phenol was observed in plants treated with salicylic acid and benzothiadiazole.

Root (wilt) disease of coconut palms in South Asia - an overview

Root (wilt) disease (RWD) caused by phytoplasma is one of the most devasting diseases of coconut palms. The major symptoms of the disease in leaves are wilting and drooping and flaccidity; ribbing, paling/yellowing and necrosis of leaflets are typical symptoms of foliar diseases. Unopened pale yellow leaflets of spindle leaves are more susceptible to leaf rot disease, which is caused by Exerohilum rostratum and Colletotrichum gloeosporioides. RWD is caused by phytoplasmas, the cell wall-less prokaryotes that are bounded by a “unit” membrane. In ultrathin sections, they appear as a complex multi-branched, beaded, filamentous or spheroidal pleomorphic bodies. The disease was transmitted by plant hoppers (Proutista moesta) and lace wing bug (Stephanitis typica). Phytoplasmas are generally present in the phloem sieve tubes and in the salivary glands of these insect vectors. Phytoplasmas cannot be cultured in vitro, and hence it is very difficult to identify them. Using polymerase chain reaction technique, group-specific primers have been applied to detect mixed-phytoplasma infections in a single host. RWD, is a non-lethal, debilitating disease, and hence an integrated approach for the management of this disease in coconut palms has been discussed in this study.

Pseudomonas fluorescens mediated systemic resistance against urdbean leaf crinkle virus in blackgram (Vigna mungo)

Abstract Studies were conducted to evaluate two specific strains of the novel plant growth promoting rhizobacterium (PGPR), Pseudomonas fluorescens for induced systemic resistance (ISR) against urdbean leaf crinkle virus (ULCV) in blackgram (Vigna mungo) under controlled conditions. Results of the studies revealed that the pre-inoculation application of blackgram plants with the strains of P. fluorescens viz., Pf1 and CHAO were found to reduce ULCV infection significantly. Soil and foliar application of P. fluorescens (Pf1) induced the accumulation of phenolics and enhanced the activities of peroxidase, phenylalanine ammonia lyase and polyphenol oxidase. New isoforms of peroxidase and polyphenol oxidase were also induced by the treatment. The induced defense mechanism might have played a role in reducing the disease.

Evaluation of antiviral principles for the induction of systemic resistance in blackgram (Vigna mungo) against Urdbean Leaf Crinkle Virus

Abstract Blackgram (Vigna mungo (L.) Hepper) is one of the important pulse crops grown all over the world. Among the virus diseases of blackgram, leaf crinkle caused by Urdbean Leaf Crinkle Virus (Ungrouped/ULCV) is an important disease which leads to considerable losses depending on the season and type of cultivar cultivated. Pre-inoculation spraying of blackgram plants with leaf extracts of Mirabilis jalapa or Bougainvillea spectabilis was found to reduce the leaf crinkle disease to about 90%. The suppression of the disease by the extract was not only due to direct inhibition of the virus. Pre-treatment of blackgram leaves with 10% leaf extracts of M. jalapa and B. spectabilis as foliar spray induced the accumulation of phenolics and enhanced the activities of peroxidase, phenylalanine ammonialyase and polyphenol oxidase. New isoforms of peroxidase and polyphenol oxidase were also induced by the treatments. The induced defense mechanism might have played a role in reducing the disease.

First report of little leaf disease associated with phytoplasma on sugar beet (Beta vulgaris L. subsp. vulgaris var. altissima Doll) in India

Sugar beet (Betavulgaris L. subsp. vulgaris var. altissima) is a biennial, sugar-producing tuber crop grown in different parts of the world. Diseased sugar beets with symptoms of little leaf were observed during 2008 in Tamil Nadu, India. Phytoplasmas were detected in symptomatic leaves of three separate plants using PCR with primer pair fU5/rU3, which amplify an 880-bp fragment of the 16S rRNA region of phytoplasmas. The nucleotide sequence analysis of a 766-bp fragment had 100% identity among the sequence from the three plants (GQ184437) and 96% nucleotide sequence identity with 16S ribosomal RNA gene sequences of eggplant phyllody phytoplasma.

Comparative Proteomic Analysis of Different Isolates of Fusarium oxysporum f.sp. lycopersici to Exploit the Differentially Expressed Proteins Responsible for Virulence on Tomato Plants

The vascular wilt of tomato caused by Fusarium oxysporum f.sp. lycopersici is an important soil borne pathogen causes severe yield loss. The molecular characterization and their interaction with its host is necessary to develop a protection strategy. 20 isolates of F. oxysporum f.sp. lycopersici (FOL) were isolated from wilt infected tomato plants across Tamil Nadu. They were subjected to cultural, morphological, molecular and virulence studies. The results revealed that all the isolates produced both micro and macro conidia with different size, number of cells. The colors of the culture and growth pattern were also varied. In addition, chlamydospores were observed terminally and intercalary. The PCR analysis with F. oxysporum species-specific primer significantly amplified an amplicon of 600 bp fragment in all the isolates. Based on the above characters and pathogenicity, isolate FOL-8 was considered as virulent and FOL-20 was considered as least virulent. Proteomics strategy was adopted to determine the virulence factors between the isolates of FOL-8 and FOL-20. The 2D analyses have showed the differential expression of 17 different proteins. Among them, three proteins were down regulated and 14 proteins were significantly up regulated in FOL-8 than FOL-20 isolate. Among the 17 proteins, 10 distinct spots were analyzed by MALDI-TOF. The functions of the analyzed proteins, suggested that they were involved in pathogenicity, symptom expression and disease development, sporulation, growth, and higher penetration rate on tomato root tissue. Overall, these experiments proves the role of proteome in pathogenicity of F. oxysporum f.sp. lycopersici in tomato and unravels the mechanism behinds the virulence of the pathogen in causing wilt disease.

Integrated Pest Management for Onion in India

Onion is one of the major commercial vegetables in India, and the main limiting factor for higher production of this crop is the incidence of pests and diseases. To reduce the pesticide treadmill, efforts were made to evaluate five different onion IPM modules at Tamil Nadu Agricultural University in India. These include the bio-intensive module comprising of selection of healthy seed bulbs, bulb treatment with Pseudomonas fluorescens and Trichoderma viride, soil amendment with biopesticides and biofertilizers, foliar application of biopesticides, and need based application of chemical pesticides. These were found to be effective in checking onion pests and diseases. The onion IPM was further fine-tuned with additional IPM components, barrier crop of maize and pheromone and sticky traps. It was demonstrated in larger fields in farm holdings of Tamil Nadu under the Integrated Pest Management Collaborative Research Support Program (IPM CRSP) and now, IPM Innovation Lab of USAID during 2009–2013 through technology transfer programs viz., demonstrations, field days, radio, farm visits, publications and others. Impact assessment on onion IPM package revealed reduced production costs, increased bulb yield, and higher economic returns.

FIRST REPORT OF ZUCCHINI YELLOW MOSAIC VIRUS ON CUCURBITA MOSCHATA IN INDIA

Zucchini yellow mosaic virus (ZYMV), genus Potyvirus, infects and causes severe yield losses to cucurbits, including Cucurbita moschata, which is widely grown and consumed in the Indian subcontinent. During 2012-2014, while conducting survey for virus diseases of cucurbits in Tamil Nadu, plants exhibiting mosaic and malformation of leaves and fruits recalling symptoms induced by ZYMV were observed in pumpkin crops at Udumalpet (Tiruppur, isolate TN UDU PUM1) and Anamalai (Coimbatore, isolate TN ANA PUM1) with incidence ranging from 40 to 70%. Ten symptomatic samples (five each from Udumalpet and Anamalai) selected randomly were tested by DAS-ELISA for the presence of ZYMV using a commercial kit (DSMZ, Germany) and ZYMV-specific immunostrips (Agdia, USA). ZYMV was detected only in symptomatic plants. Likewise, RT-PCR using the primer pair (GK ZYMV F/R2) constructed on the coat protein sequence of the virus (Nagendran et al., 2015) amplified a product of ca. 1,000 bp only from symptomatic samples, which was cloned and sequenced in both orientations. Multiple alignment of the nucleotide sequences of both viral isolates (KJ866937 and KJ729043) with comparable sequences from GenBank revealed 98-99% identity with ZYMV isolates from India (HQ529776, KJ866939, KJ729041, KJ729044, JF797206), Iran (JN183062), Israel (EF062582), Syria (KF056805) and Germany (AJ420019) confirming the presence of this virus in symptomatic pumpkin plants. The occurrence of ZYMV in pumpkin has been reported from Germany, Serbia, Korea and Australia (Vucurovic et al. 2012), and in cucumber, gherkins, snake gourd, bottle gourd, zucchini and Amaranthus viridis in India (Nagendran et al., 2015). This is then the first report of ZYMV occurrence in pumpkin in India.

Molecular characterization and in vitro evaluation of endophytic bacteria against major pathogens of rice

Lallemantia royleana (Benth.) is an important folk medicine in Pakistan. This natural herb is used as folk remedy for number of ailments. This study was conducted to assess the antibacterial activity of four different organic extracts of L. royleana seeds against four bacterial strains (Escherichia coli, Enterobacter cloaceae, Pseudomonas aeruginosa and Staphylococcus aureus) for the first time by disc diffusion method. Except aqueous extracts, all organic extracts of L. royleana seeds displayed significant anti-bacterial activity against all the tested bacteria. The chloroform extract exhibited highest anti-bacterial activity for all bacterial strains. Results shows that L. royleana seeds possess significant antibacterial potential against S. aureus, E. coli and E. cloaceae, therefore, it can be a good remedy for skin disease and gastro-intestinal problems caused by human pathogenic bacterial strains. Further screening for phytochemicals should be carried out in search of novel therapeutic compounds.

Production of polyclonal antibodies for Capsicum chlorosis virus (CaCV) infecting chilli in India through recombinant nucleocapsid protein expression and its application

Bud necrosis and chlorotic spots causing virus affecting chilli crop in Tamil Nadu (India) was identified as Capsicum chlorosis virus (CaCV). Specific primers were used for amplification and sequencing of the nucleocapsid protein (NP) gene. Polyclonal antibody against the bacterially expressed NP from the CaCV-TN-CBE isolate was produced using recombinant DNA technology. NP gene was subcloned into the pET-28a (+) vector and expressed by transformation in BL21 (DE3) pLysS. The expressed protein was about ∼34 kDa and was confirmed through western blot analysis using Groundnut bud necrosis virus (GBNV) polyclonal antiserum from ICRISAT, India. The purified recombinant protein was used to immunize rabbits to generate CaCV-specific polyclonal antiserum. The sensitivity levels of polyclonal antiserum thus raised was assayed through indirect ELISA or direct antigen coating (DAC)-ELISA using the recombinant protein as antigen. The recombinant antiserum produced in this study successfully detected the natural infection of CaCV on chilli plants collected from the field as well as on cowpea plants artificially inoculated with CaCV by using DAC-ELISA, DIBA and western blotting.