A survey of die-back disease of neem in Tamil Nadu,India and PCR-based confirmation of the isolates

A survey of die-back disease of neem was done in different agro climatic regions of Tamil Nadu, India using Global Positioning System (GARMIN 12). Twigs of Azadirachta indica (Neem) infected with die-back were collected from different regions of Tamil Nadu, India and they were further analyzed to determine the pathogen. Phomopsis azadirachtae the causal organism was isolated on malt extract agar from die-back infected neem twigs. They were identified by conventional and molecular methods. Phomopsis genus specific primers (5.8S r-DNA) were then used for the confirmation of P. azadirachtae – the causative agent of die-back of neem by Polymerase chain reaction (PCR). Studies revealed the amplification of expected 141bp DNA in P. azadirachtae isolated from the diseased trees of different regions of Tamil Nadu confirming the causal organism of die-back of neem. Studies revealed a very high incidence of die-back in most of the places of Tamil Nadu. Hand held GPS was used in the study which would help in continuous monitoring of the diseased trees.     

Determination of biotic aetiology of tapping panel dryness (TPD) syndrome of rubber tree (Hevea brasiliensis) by return-polyacrylamide gel electrophoresis (R-PAGE) technique

Tapping panel dryness (TPD) syndrome affecting rubber tree (Hevea brasiliensis) is known to reduce natural latex production. Its aetiology remains ambiguous despite long years of research. A low molecular weight RNA similar to viroid RNA was isolated from TPD-affected samples of rubber trees. In the present study, a modified return-polyacrylamide gel electrophoresis procedure was standardised. The viroid-like low molecular weight (LMW) RNA was found associated with leaf, bark and root tissues and rubber seedlings. The technique was employed to detect LMW RNA in different clones of rubber planted in different locations and in bud-grafted plants. The LMW RNA isolated from TPD-affected trees was found infectious on seedlings of tomato cv Pusa Ruby. The LMW RNA was reisolated from symptomatic tomato leaves but not from control plants. This is for the first time that a biotic agent, a viroid RNA, is found consistently associated with the syndrome. The technology developed can be useful to demonstrate the onset of TPD in untapped trees in the absence of other methods such as nucleic acid hybridisation.     

The effect of Meloidogyne incognita on the histopathology of Momordica charantia roots

Bitter gourd (Momordica charantia L.) was inoculated with root-knot nematode Meloidogyne incognita to investigate the anatomical abnormalities in the affected roots. Soon after inoculation the second-stage juveniles (J2) entered at or near the root caps and migrated intercellularly towards the zone of vascular differentiation. Discrete giant cells were observed after three days of inoculation. The nematode induced hypertrophy and hyperplasia near the giant cells. After six days, the juveniles moulted to their third stage (J3). At the same, time giant cell size and density of giant cell cytoplasm increased. The continuity of vascular strands remained unaffected. Between 12 and 24 days of inoculation the giant cells enlarged several times and became multinucleate and enclosed dense and granular cytoplasm. The nematodes became almost pyriform 18 days after inoculation. The orientation of vascular strands changed, due to hypertrophy, hyperplasia and enlargement of the nematode. After 30 days of inoculation the nematodes developed into mature females and started egg laying. A large amount of parenchyma transformed into abnormal xylem.     

Rice Resistance to Planthoppers and Leafhoppers

For over 50 years, host-plant resistance has been regarded as an efficient method to reduce yield losses to rice caused by delphacid and cicadelid hoppers. Already a number of resistant rice varieties have been developed and deployed throughout Asia. To date, over 70 hopper resistance genes have been identified in rice; however, less than 10 genes have been deliberately introduced to commercial rice varieties. Currently, due to recent brown planthopper (Nilaparvata lugens [Stål]) and whitebacked planthopper (Sogatella furcifera [Horvath]) outbreaks occurring at an unprecedented scale, researchers are working toward a second generation of resistant varieties using newly identified gene loci and applying new molecular breeding methods. This paper reviews advances in the identification of resistance genes and QTLs against hoppers in rice. It collates all published information on resistance loci and QTLs against the major rice planthoppers and leafhoppers and presents information on gene locations, genetic markers, differential varieties, and wild rice species as sources of resistance. The review indicates that, whereas progress in the identification of genes has been rapid, considerable tidying of the information is required, especially regarding gene nomenclature and resistance spectra. Furthermore, sound information on gene functioning is almost completely lacking. However, hopper responses to resistance mechanisms are likely to be similar because a single phenotyping technique has been applied by most national and international breeding programs during germplasm screening. The review classifies genes occurring at two chromosome regions associated with several identified resistance loci and highlights these (Chr4S: BphR-R and Chr12L: BphR-R) as general stress response regions. The review calls for a greater diversity of phenotyping methods to enhance the durability of resistant varieties developed using marker-aided selection and emphasizes a need to anticipate the development of virulent hopper populations in response to the field deployment of genes.     

Topology,glycosylation and conformational changes in the membrane domain of the vacuolar H+‐ATPase a subunit

Published topological models of the integral membrane a subunit of the vacuolar proton‐translocating ATPase complex have not been in agreement with respect to either the number of transmembrane helices within the integral membrane domain, or their limits and orientations within the lipid bilayer. In the present work we have constructed a predictive model of the membrane insertion of the yeast a subunit, Vph1p, from a consensus of seven topology prediction algorithms. The model was tested experimentally using epitope tagging, green fluorescent protein fusion, and protease accessibility analysis in purified yeast vacuoles. Results suggest that a consensus prediction of eight transmembrane helices with both the amino‐terminus and carboxyl‐terminus in the cytoplasm is correct. Characterization of two glycosylation sites within the homologous mouse a subunit membrane domain further corroborates this topology. Moreover, the model takes into account published data on cytoplasmic and luminal accessibility of specific amino acids. Changes in the degree of protease accessibility in response to the V‐ATPase substrate, MgATP, and the V‐ATPase‐specific inhibitor, concanamycin A, suggest that functional conformational changes occur in the large cytoplasmic loop between TM6 and TM7 of Vph1p. These data substantially confirm one topological model of the V‐ATPase a subunit and support the notion that conformational changes occur within the membrane domain, possibly involving previously proposed axial rotation and/or linear displacement of TM7 in the proton transport cycle. J. Cell. Biochem. 114: 1474–1487, 2013. © 2013 Wiley Periodicals, Inc.     

Abelmoschus enbeepeegearense sp. nov. (Malvaceae), an endemic species of okra from Western Ghats,India

Abelmoschus enbeepeegearense J. John et al. is a new species occurring at low elevations in the Western Ghats of India, comprising Kerala, Karnataka and Tamil Nadu. The taxon is morphologically allied to A. moschatus subsp. moschatus, A. moschatus subsp. tuberosus and A. crinitus, but easily distinguishable by virtue of its orthotropic branching, 3–5‐angled leaves, glandular hairy plant body with whitish waxy secretions, glandular non‐setose epiclayx segmens which is more than eight in number and ovate hirsute fruits with a short mucro at the apex. It can be crossed with all three taxa with varying degree of success, but the hybrids are sterile. The material belonging to it was earlier identified as and placed under A. moschatus Medik. The taxon is described and illustrated with notes on its phenology, ecology and distribution. In additions, a key to all Abelmoschus taxa occurring in India is provided.     

Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains

A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.     

Insight into Pleiotropic Drug Resistance ATP-binding Cassette Pump Drug Transport through Mutagenesis of Cdr1p Transmembrane Domains

The fungal ATP-binding cassette (ABC) transporter Cdr1 protein (Cdr1p), responsible for clinically significant drug resistance, is composed of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). We have probed the nature of the drug binding pocket by performing systematic mutagenesis of the primary sequences of the 12 transmembrane segments (TMSs) found in the TMDs. All mutated proteins were expressed equally well and localized properly at the plasma membrane in the heterologous host Saccharomyces cerevisiae, but some variants differed significantly in efflux activity, substrate specificity, and coupled ATPase activity. Replacement of the majority of the amino acid residues with alanine or glycine yielded neutral mutations, but about 42% of the variants lost resistance to drug efflux substrates completely or selectively. A predicted three-dimensional homology model shows that all the TMSs, apart from TMS4 and TMS10, interact directly with the drug-binding cavity in both the open and closed Cdr1p conformations. However, TMS4 and TMS10 mutations can also induce total or selective drug susceptibility. Functional data and homology modeling assisted identification of critical amino acids within a drug-binding cavity that, upon mutation, abolished resistance to all drugs tested singly or in combinations. The open and closed Cdr1p models enabled the identification of amino acid residues that bordered a drug-binding cavity dominated by hydrophobic residues. The disposition of TMD residues with differential effects on drug binding and transport are consistent with a large polyspecific drug binding pocket in this yeast multidrug transporter.