First Report of Tobacco streak virus Infecting Pigeon Pea (Cajanus cajan) in India.

Pigeon pea is an important pulse crop grown in diversified cropping systems in India. In the rainy season of August 2011 and September 2012, pigeon pea cv. LRG 30 plants with leaf necrosis having wrinkled margin on one side were observed in Kadiri mandal of the Anantapuram district of Andhra Pradesh (A.P.), India. Symptoms included necrotic spots on young leaves followed by wilting of leaves, petiole and branch/axillary shoot proliferation, with small leaves having mosaic symptoms. Symptomatic leaves were sap-inoculated onto 10 seedlings of cowpea (cv. Pusa Komal) using 0.01 M phosphate buffer (pH 7.0). Localized necrotic lesions developed in all the inoculated plants after 2 days post inoculation. Field symptoms were reproduced on healthy pigeon pea upon back inoculation using single lesions of infected cowpea leaves. In direct antigen coating (DAC)-ELISA, all the infected pigeon pea and cowpea leaf samples were positive to a polyclonal antiserum specific to Tobacco streak virus (TSV) supplied by ICRISAT, India. Total RNA was extracted using infected pigeon pea and healthy leaf samples by TRI Reagent (Sigma). Reverse transcription (RT)-PCR was carried out using primers specific to the coat protein (CP) gene of TSV (1). A product of the 700-bp DNA fragment was obtained in field-infected pigeon pea samples but not in healthy controls. The amplicon was cloned into PTZ57R/T using the Ins TA clone PCR kit (Fermentas). Recombinant clone was sequenced in both directions and the CP gene sequence obtained was deposited in GenBank (KF220492). Sequence analysis of the CP gene of TSV from pigeon pea shared 98 to 100% identity with Indian TSV isolates originating from different hosts including groundnut (FJ355948), mung bean (FJ749259), and sunflower (DQ864448), and 88 to 92% similarity with TSV type isolate (white clover: X00435) both at nucleotide and amino acid levels. TSV belongs to the genus Ilarvirus of family Bromoviride and has a wide host range. TSV is pollen borne, assisted by thrips causing mechanical injury (2). To our knowledge, this is the first report of TSV on pigeon pea in India and was widespread in Anantapuram, Kadapa, Kurnool, and Mahbubnagar districts of A.P. Yield loss depends on the stage of infection as early infection resulted in complete failure of the crop. TSV was prevalent on many legume crops such as black gram, green gram, and groundnut in A.P, Tamil Nadu, Karnataka, and Maharashtra states (3). TSV infection of pigeon pea may pose a serious implication for pulse production. References: (1). A. I. Bhat et al. Arch. Virol. 147:651, 2002. (2). M. Sharman et al. Australian Plant Dis. 6:54, 2011. (3). K. Vemana and R. K. Jain. Indian J. Virol. 21:117, 2010.

Determination of antibiotics in aquatic environment by solid-phase extraction followed by liquid chromatography-electrospray ionization mass spectrometry.

A robust and sensitive solid-phase extraction followed by liquid chromatography-electrospray ionization mass spectrometric (LC-ESI-MS) method for determination of antibiotics viz., fluoroquinolones, sulfamethoxazole, trimethoprim and cephalosporines in surface waters was developed. The sample recoveries on Oasis HLB cartridges were found to be >80%. Identification was carried out by LC-ESI-MS/MS. The positive ion ESI mass spectra containing the peaks of quasimolecular ions [M+H](+) allowed the determination of molecular masses whereas the fragment ions obtained by MS/MS of [M+H](+) ions permitted the structural assignment. Quantification was carried out by selective ion monitoring (SIM) using the quasimolecular ions [M+H](+) of the parent compounds. The detection and quantification limits were found to be in the range of 0.6-8.1 and 2.0-24.0 microg/L. The surface waters of different lakes and tanks of Hyderabad, India were found to contain a few antibiotics.

Use of solid-phase extraction, reverse osmosis and vacuum distillation for recovery of aromatic sulfonic acids from aquatic environment followed by their determination using liquid chromatography-electrospray ionization tandem mass spectrometry.

Three different sample preparation techniques (i) solid-phase extraction, (ii) reverse osmosis and (iii) vacuum distillation have been investigated and the recoveries were compared for determination of highly water-soluble benzene and stilbene sulfonic acids in aqueous environment by liquid chromatography with photodiode array (PDA) and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The recoveries were quite high using vacuum distillation (>90%) compared to solid phase extraction and reverse osmosis. The negative ion ESI mass spectra containing the peaks of quasimolecular ion [M-H]- allow the molecular mass determination of unknown compounds whereas the structures were proposed using fragments obtained from MS/MS analysis of [M-H]- ions. At lower fragmentation voltages only the quasimolecular ion [M-H]- was observed and as fragmentation voltages increased, it led to the formation of fragment ions corresponding to [M-H-SO3]-, [M-H-SO2]-, and SO3-. The detection limits were 1-28 microg/L with LC-ESI-MS. The sample collected from wastewater treatment plant was found to contain 21.1, 13.3, 12.1, 41.8 and 9.9 microg/L of cis-4,4(l)-diaminostilbene-2,2(l)-disulfonic acid (cis-DASDA), trans-4,4(l)-diaminostilbene-2,2(l)-disulfonic acid (trans-DASDA), 3-amino acetanilide-4-sulfonic acid (3-AASA), 4-chloroaniline-2-sulfonic acid (4-CASA), 2-chloroaniline-5-sulfonic acid (2-CASA), respectively.

LC-PDA and LC-ESI-MS separation and determination of process-related substances arising from stilbene-type fluorescent whitening agents. Application to monitoring of their photodegradation products in industrial effluents and aqueous environmental systems.

A simple and rapid gradient elution high-performance liquid chromatographic method using photodiode array and electrospray ionization mass spectrometric detectors was developed for separation and determination of the process-related substances and photodegradation products of stilbenesulfonic acids, viz. 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNSDA), 4-amino-4'-nitrostilbene-2,2'-disulfonic acid (ANSDA), and 4,4'-diaminostilbene-2,2'-disulfonic acid (DASDA) in industrial waste waters. Gradient elution was carried out using ammonium acetate and acetonitrile as mobile phase and an Inertsil-ODS 3V column for separation. The negative-ion electrospray ionization mass spectra containing [M-H]- ions of sulfonic acids allowed molecular mass determination of unknowns and the structures were proposed on the basis of the fragment ions in the MS/MS spectra.

Development and validation of a liquid chromatographic method for monitoring of process-related synthetic organic impurities of profenofos in technical products.

A simple and rapid reversed-phase high-performance liquid chromatographic method for the monitoring of process-related synthetic organic impurities of profenofos (PFS) is developed. Impurities are separated and determined on a reversed-phase Hypersil C(18) column using gradient elution of 50 mM ammonium formate buffer-acetonitrile as a mobile phase and detection at 230 nm at ambient temperature. The method is validated with respect to accuracy, precision, linearity, and limits of detection and quantitation. The method is found to be suitable not only for monitoring the reactions involved in the process development of PFS, but also quality assurance, as it can detect impurities at the level of 1.5 x 10(-8) g.

Liquid chromatographic separation and determination of aromatic sulfonates in an aquatic environment using a photodiode array and electrospray ionization-mass spectrometer as detectors.

A simple and rapid method involving high-performance liquid chromatographic separation, followed by photodiode array (PDA) and electrospray ionization-mass spectrometric (ESI-MS) detection of aromatic sulfonates in waste-water effluents of industrial units producing optical whitening agents, has been developed. The separation was achieved on a reversed-phase Hypersil C18 column using gradient elution of a mobile phase consisting of 0.05 M ammonium formate-methanol with decreasing concentration of the buffer at room temperature. The minimum detection limits were determined to be in the range of 0.2 - 1.8 x 10(-9) g using PDA and ESI-MS detectors.