PHYLOGENETIC ANALYSIS OF RED COTTON BUG SPECIES (HEMIPTERA: PYRRHOCORIDAE) IN PUNJAB, PAKISTAN

Traditional techniques for identifying the red cotton bugs Dysdercus koenigii and Dysdercus cingulatus (Pyrrhocoridae: Hemiptera) depend on phenotypic and morphological data. These methods are suitable when species have well-defined structures and when huge taxonomic knowledge is at hand. In this study, the mitochondrial cytochrome oxidase I (COI) gene was selected for the molecular identification of D. koenigii and D. cingulatus. The sequence data showed that one species of the red cotton bug sample was identified as Dysdercus koenigii with 35-100% similarity to other Dysdercus species present in the NCBI database, while the other species was identified as Dysdercus cingulatus with 88-94% similarity to other red cotton bugs species present in the NCBI database. The phylogenetic analysis revealed that our     D. koenigii Seq (>180319003-A02-102-DK-.ab1) shares the same cluster with four D. koenigii isolates (ZSI/SRC_I.28B, ZSI/SRC_I.28, GQ306227.1, KJ459924.1). While the D. cingulatus Seq (>180319003-A02-103-DC-.abl) shares a cluster with D. cingulatus voucher (RO_DC2015). This is the first molecular identification of red cotton bugs (D. koenigii and     D. cingulatus) reported in Pakistan. Thus, in the current study, molecular identification has been accepted as a reliable method for the identification of these agriculturally important insect pests

Cytotoxicity against A549 Human Lung Cancer Cell Line via the Mitochondrial Membrane Potential and Nuclear Condensation Effects of Nepeta paulsenii Briq., a Perennial Herb

The genus Nepeta belongs to the largest Lamiaceae family, with 300 species, which are distributed throughout the various regions of Africa, Asia, India, and America. Along with other plant families distinguished by their medicinal and therapeutic values, the Nepeta genus of Lameaceae remains relatively valuable. Hence, the phytochemicals of N. paulsenii Briq. were extracted using different plant parts, i.e., leaves, stem, roots, flowers, and the whole plant by using various solvents (ethanol, water, and ethyl acetate), obtaining 15 fractions. Each extract of dried plant material was analyzed by FT-IR and GC-MS to identify the chemical constituents. The cytotoxicity of each fraction was analyzed by MTT assay and mitochondrial membrane potential and nuclear condensation assays against lung cancer cells. Among the ethyl acetate and ethanolic extracts, the flowers showed the best results, with IC50 values of 51.57 μg/mL and 50.58 μg/mL, respectively. In contrast, among the water extracts of the various plant segments, the stem showed the best results, with an IC50 value of 123.80 μg/mL. 5-flourouracil was used as the standard drug, providing an IC50 value of 83.62 μg/mL. The Hoechst 33342 stain results indicated apoptotic features, i.e., chromatin dissolution and broken down, fragmented, and crescent-shaped nuclei. The ethanolic extracts of the flowers showed more pronounced apoptotic effects on the cells. The mitochondrial membrane potential indicated that rhodamine 123 fluorescence signals suppressed mitochondrial potential due to the treatment with the extracts. Again, the apoptotic index of the ethanolic extract of the flowers remained the highest. Hence it can be concluded that the flower part of N. paulsenii Briq. was found to be the most active against the A459 human lung cancer cell line

Etude de l'expression de gènes impliqués dans les voies de défense et les mécanismes épigénétiques chez la tomate infectée par le phytoplasme du stolbur

Les phytoplasmes sont des bactéries phytopathogènes, sans paroi, qui appartenant à la classe des Mollicutes. Ils ne peuvent pas etre cultivés in vitro et sont limités à des tubes du phloème. Ils provoquent des centaines de maladies chez de nombreuses espèces végétales dans le monde entier, ce qui conduit à des pertes de récolte importantes. Les phytoplasmes sont transmis naturellement par des insectes suceurs de sève dans laquelle ils se multiplient. Ils induisent des symptômes graves, notamment le jaunissement, la croissance limitée, déclin, ainsi que des anomalies des fleurs et des fruits. L'infection par le phytoplasme du stolbur, en particulier, affect fortement la morphologie florale. Dans la tomate, deux isolats différents du phytoplasme du stolbur, nommé C et PO, induisent des symptômes différents. La tomate infectée par le phytoplasme du stolbur PO montrent des malformations florale telles que les sépales hypertrophiés, les pétales et les étamines avortées ce qui conduit à la stérilité. En revanche, la tomate infecté par le phytoplasme du stolbur C ont de petites feuilles de tomate en retrait, mais les fleurs presque normale, et produisent des fruits. Nous avons précédemment montré que SlDEF, un gène impliqué dans la formation des pétales est réprimé dans des plantes de tomate infectée par le stolbur phytoplasme PO. Toutefois, l'expression de son facteur de transcription, codée par le gène FA, est resté stable ou voir légèrement augmentée. Nous avons donc émis l'hypothèse que la répression de SlDEF pourrait être dû à une méthylation de l'ADN. Pour tester cette hypothèse, nous avons étudié l'expression des gènes de méthylases et de déméthylases. Ils étaient en général réprimés dans les tomates infectées par le phytoplasme du stolbur PO, ce qui était en accord avec l'hypothèse De plus, nous avons étudié les voies de défense activée chez les tomates infectées par le phytoplasme du stolbur. Pour se défendre, les plantes utilisées des molécules de signalisation comme l'acide salicylique (SA), l'acide jasmonique (JA) et d'éthylène (ET). Nous avons étudié l'expression de 21 gènes de défense dépendants SA / JA / ET, des gènes de biosynthèse et les facteurs de transcription chez les tomates infectées par les phytoplasmes du stolbur C et PO. Nous avons également étudié l'effet de la pré-activation des voies de SA et JA sur la production des symptômes. Nos résultats montrent clairement que les voies de défense ont été activées différemment dans les tomates infectés par le phytoplasme du stolbur C et PO. En effet, les voies de défense dépendantes de SA, ET et JA ont été activées chez les tomates infectées par le phytoplasme du stolbur C alors que seulement les voies dépendantes SA et ET ont été activés dans les tomates infectées par stolbur PO . En outre, la pré-activation de la voie de défense dépendante SA par l'application de BTH modifie légèrement l'évolution des symptômes de maladies causées par le phytoplasme du stolbur POPhytoplasma are cell wall-less, phytopathogenic bacteria belonging to the class Mollicutes. They have not been cultured in vitro and are restricted to the phloem sieve tubes. They cause hundreds of diseases in many plant species worldwide, resulting in important crop losses. Phytoplasmas are naturally transmitted by sap-sucking insects in which they multiply. They induce severe symptoms including yellowing, restricted growth, decline, as well as major flowers and fruits abnormalities.The stolbur phytoplasma infection, in particular, has been reported to strongly affect floral morphology. In tomato, two different isolates of stolbur phytoplasma, named C and PO, induce different symptoms. The stolbur PO phytoplasma-infected plants show abnormal flower development such as hypertrophied sepals, and aborted petals and stamens leading to sterility. In contrast, stolbur C phytoplasma-infected tomato have small indented leaves but nearly normal flowers, and produce fruits. We have previously shown that SlDEF, one gene involved in petal formation, was repressed in stolbur PO phytoplasma-infected tomato. However, the expression of its transcription factor, encoded by the gene FA, was unchanged or slightly up-regulated. So we hypothesized that SlDEF repression could be due to DNA methylation. To test this hypothesis, we studied the expression of DNA methylases and demethylases genes. They were in general down-regulated in stolbur PO infected tomato, which was in agreement with the hypothesis. However, the regulation of SlDEF expression could not be firmly correlated to the DNA methylation status of its promoter region. In addition, we studied the plant defense pathways activated in stolbur phytoplasma-infected tomato. To defend themselves, plants used signalling molecules like Salicylic acid (SA), Jasmonic acid (JA) and Ethylene (ET). We studied the expression of 21 SA/JA/ET regulated defense and biosynthesis genes including transcription factors in stolbur C and PO phytoplasma-infected tomato as compared to healthy ones. We also studied the effect of pre-activation of SA and JA mediated defense pathways on symptom production. Our results clearly showed that defense pathways were activated differently in stolbur C and PO phytoplasma-infected tomato. Indeed, SA ET and JA dependant pathways were activated in stolbur C-infected tomato while only SA and ET dependant pathways were activated in stolbur PO-infected plants. In addition, pre-activation of SA-dependent defense pathway by application of BTH slightly modify the evolution of disease symptoms caused by stolbur PO phytoplasma whereas no effect was observed after treatment with an analogue of JA

Study of the Expression of Genes involved in Defense pathways and Epigenetic Mechanisms in tomato infected with Stolbur Phytoplasma

Les phytoplasmes sont des bactéries phytopathogènes, sans paroi, qui appartenant à la classe des Mollicutes. Ils ne peuvent pas etre cultivés in vitro et sont limités à des tubes du phloème. Ils provoquent des centaines de maladies chez de nombreuses espèces végétales dans le monde entier, ce qui conduit à des pertes de récolte importantes. Les phytoplasmes sont transmis naturellement par des insectes suceurs de sève dans laquelle ils se multiplient. Ils induisent des symptômes graves, notamment le jaunissement, la croissance limitée, déclin, ainsi que des anomalies des fleurs et des fruits. L'infection par le phytoplasme du stolbur, en particulier, affect fortement la morphologie florale. Dans la tomate, deux isolats différents du phytoplasme du stolbur, nommé C et PO, induisent des symptômes différents. La tomate infectée par le phytoplasme du stolbur PO montrent des malformations florale telles que les sépales hypertrophiés, les pétales et les étamines avortées ce qui conduit à la stérilité. En revanche, la tomate infecté par le phytoplasme du stolbur C ont de petites feuilles de tomate en retrait, mais les fleurs presque normale, et produisent des fruits. Nous avons précédemment montré que SlDEF, un gène impliqué dans la formation des pétales est réprimé dans des plantes de tomate infectée par le stolbur phytoplasme PO. Toutefois, l'expression de son facteur de transcription, codée par le gène FA, est resté stable ou voir légèrement augmentée. Nous avons donc émis l'hypothèse que la répression de SlDEF pourrait être dû à une méthylation de l'ADN. Pour tester cette hypothèse, nous avons étudié l'expression des gènes de méthylases et de déméthylases. Ils étaient en général réprimés dans les tomates infectées par le phytoplasme du stolbur PO, ce qui était en accord avec l'hypothèse De plus, nous avons étudié les voies de défense activée chez les tomates infectées par le phytoplasme du stolbur. Pour se défendre, les plantes utilisées des molécules de signalisation comme l'acide salicylique (SA), l'acide jasmonique (JA) et d'éthylène (ET). Nous avons étudié l'expression de 21 gènes de défense dépendants SA / JA / ET, des gènes de biosynthèse et les facteurs de transcription chez les tomates infectées par les phytoplasmes du stolbur C et PO. Nous avons également étudié l'effet de la pré-activation des voies de SA et JA sur la production des symptômes. Nos résultats montrent clairement que les voies de défense ont été activées différemment dans les tomates infectés par le phytoplasme du stolbur C et PO. En effet, les voies de défense dépendantes de SA, ET et JA ont été activées chez les tomates infectées par le phytoplasme du stolbur C alors que seulement les voies dépendantes SA et ET ont été activés dans les tomates infectées par stolbur PO . En outre, la pré-activation de la voie de défense dépendante SA par l'application de BTH modifie légèrement l'évolution des symptômes de maladies causées par le phytoplasme du stolbur POPhytoplasma are cell wall-less, phytopathogenic bacteria belonging to the class Mollicutes. They have not been cultured in vitro and are restricted to the phloem sieve tubes. They cause hundreds of diseases in many plant species worldwide, resulting in important crop losses. Phytoplasmas are naturally transmitted by sap-sucking insects in which they multiply. They induce severe symptoms including yellowing, restricted growth, decline, as well as major flowers and fruits abnormalities.The stolbur phytoplasma infection, in particular, has been reported to strongly affect floral morphology. In tomato, two different isolates of stolbur phytoplasma, named C and PO, induce different symptoms. The stolbur PO phytoplasma-infected plants show abnormal flower development such as hypertrophied sepals, and aborted petals and stamens leading to sterility. In contrast, stolbur C phytoplasma-infected tomato have small indented leaves but nearly normal flowers, and produce fruits. We have previously shown that SlDEF, one gene involved in petal formation, was repressed in stolbur PO phytoplasma-infected tomato. However, the expression of its transcription factor, encoded by the gene FA, was unchanged or slightly up-regulated. So we hypothesized that SlDEF repression could be due to DNA methylation. To test this hypothesis, we studied the expression of DNA methylases and demethylases genes. They were in general down-regulated in stolbur PO infected tomato, which was in agreement with the hypothesis. However, the regulation of SlDEF expression could not be firmly correlated to the DNA methylation status of its promoter region. In addition, we studied the plant defense pathways activated in stolbur phytoplasma-infected tomato. To defend themselves, plants used signalling molecules like Salicylic acid (SA), Jasmonic acid (JA) and Ethylene (ET). We studied the expression of 21 SA/JA/ET regulated defense and biosynthesis genes including transcription factors in stolbur C and PO phytoplasma-infected tomato as compared to healthy ones. We also studied the effect of pre-activation of SA and JA mediated defense pathways on symptom production. Our results clearly showed that defense pathways were activated differently in stolbur C and PO phytoplasma-infected tomato. Indeed, SA ET and JA dependant pathways were activated in stolbur C-infected tomato while only SA and ET dependant pathways were activated in stolbur PO-infected plants. In addition, pre-activation of SA-dependent defense pathway by application of BTH slightly modify the evolution of disease symptoms caused by stolbur PO phytoplasma whereas no effect was observed after treatment with an analogue of JA

Study of the expression of defense related protein genes in stolbur C and stolbur PO phytoplasma-infected tomato

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699International audiencePhytoplasma are phloem-restricted plant pathogenic bacteria that cause hundreds of diseases. They are not cultivated in vitro and can be transmitted through insect vectors or grafting. Researches have been investigating defense pathways to different pathogens (bacteria, fungi...) but little is known about defense pathways activated in phytoplasma infected plants. In this study, the expression of genes related to salicylic acid (SA), ethylene (ET) and jasmonic acid (JA) pathways have been investigated in stolbur C phytoplasma and stolbur PO phytoplasma-infected tomato. We noted elevated level of expression for Pathogenesis Related (PR) genes. Transcription of SA and ET marker genes was up-regulated in stolbur PO and stolbur C-infected tomato, while transcription of PIN2, a gene regulated by JA, increased only in stolbur C-infected tomato

Study of the expression of defense related protein genes in stolbur C and stolbur PO phytoplasma-infected tomato

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699International audiencePhytoplasma are phloem-restricted plant pathogenic bacteria that cause hundreds of diseases. They are not cultivated in vitro and can be transmitted through insect vectors or grafting. Researches have been investigating defense pathways to different pathogens (bacteria, fungi...) but little is known about defense pathways activated in phytoplasma infected plants. In this study, the expression of genes related to salicylic acid (SA), ethylene (ET) and jasmonic acid (JA) pathways have been investigated in stolbur C phytoplasma and stolbur PO phytoplasma-infected tomato. We noted elevated level of expression for Pathogenesis Related (PR) genes. Transcription of SA and ET marker genes was up-regulated in stolbur PO and stolbur C-infected tomato, while transcription of PIN2, a gene regulated by JA, increased only in stolbur C-infected tomato

Molecular Identification and Pathological Characteristics of NPV Isolated from Spodoptera litura (Fabricius) in Pakistan

The cotton army worm Spodoptera litura Fabricius (Lepidoptera: Noctuidae) is a destructive pest of various field crops and vegetables in Pakistan. Development of biopesticide is an attractive strategy to minimize the problems of pest resistance, environmental pollution and human health concerns. The isolates of S. litura nucleopolyhedroviruses (SltNPV) were collected from infected larvae fed on natural cotton crops. The NPV was isolated from the larvae and viral occlusion bodies (VOBs) were detected using a light microscope. The toxicity of native isolates against S. litura also studied by testing different concentrations (1 x 102 POB (Polyhedral occlusion bodies) mL-1- 1 x 1010 POB mL-1) from the occlusion bodies produced from NPV isolates against 2nd, 3rd, 4th and 5th instar larvae of S. litura. The rapid and sensitive polymerase chain reaction (PCR) technique was used for the molecular detection of NPV gene from native NPV diseased insect. Multiple sequence alignment and phylogenetic analysis were performed to compare SlNPV- FSD15 based on Lef-8 with other Lef-8 genes sequences clearly showed that our SlNPV-FSD15 isolate belongs to Spodoptera litura associated NPVs. The biological activities of this NPV isolates were investigated under laboratory condition. The highest mortality of S. litura was observed at early instars. Against second instars of S. litura, LC50 values of NPV isolate ranged from 1.92×103 to 3.64×103OB/ml with LT50 values of 69.30 h to 72.80 h, respectively. This study showed highly effectiveness and provides an opportunity to cut down the use of synthetic approaches and develop safe biological/microbial insecticides from NPV isolates, which in future may effectively control S. litura

Expression of defence genes in stolbur phytoplasma infected tomatoes, and effect of defence stimulators on disease development

In tomato, the stolbur disease caused by ‘Candidatus Phytoplasma solani’ alters developmental processes resulting in malformations of both vegetative and reproductive organs, two stolbur phytoplasma strains PO and C induce mutually distinct symptoms. The aim of the present study was to determine the effect of stolbur phytoplasma-infection on the Salicylic (SA) and Jasmonic (JA) acids hormone signalling pathways and to assess whether pre-activation of these defence pathways could protect tomato against the stolbur disease development. Expression of SA- and JA-dependent marker genes was studied in tomato by qRT-PCR. Results indicated that the SA-mediated defence response was activated by the stolbur phytoplasma strains PO and C in contrast to the JA-dependent defence pathway which was repressed by strain PO but activated by strain C. The two stolbur strains, PO and C, generated different responses, suggesting that the two strains might have distinct virulence factors, in agreement with the fact that they induce distinctive symptoms. In stolbur PO-infected tomato, pre-activation of the JA-dependent defence pathway by methyl jasmonate (MeJA) before infection had no effect on the disease development whereas pre-activation of the SA-dependent defence pathway by treatment with benzothiadiazole (BTH) prior to graft-inoculation of the phytoplasma resulted in a minor delay in phytoplasma multiplication and symptom production. As grafting implicates a high inoculum as compared to insect inoculation, it would be of interest to test BTH treatment in natural conditions

The Imaging of Guard Cells of <i>thioglucosidase</i> (<i>tgg</i>) Mutants of Arabidopsis Further Links Plant Chemical Defence Systems with Physical Defence Barriers

The glucosinolate-myrosinase system is a well-known plant chemical defence system. Two functional myrosinase-encoding genes, THIOGLUCOSIDASE 1 (TGG1) and THIOGLUCOSIDASE 2 (TGG2), express in aerial tissues of Arabidopsis. TGG1 expresses in guard cells (GCs) and is also a highly abundant protein in GCs. Recently, by studying wild type (WT), tgg single, and double mutants, we showed a novel association between the glucosinolate-myrosinase system defence system, and a physical barrier, the cuticle. In the current study, using imaging techniques, we further analysed stomata and ultrastructure of GCs of WT, tgg1, tgg2 single, and tgg1 tgg2 double mutants. The tgg mutants showed distinctive features of GCs. The GCs of tgg1 and tgg1 tgg2 mutants showed vacuoles that had less electron-dense granular material. Both tgg single mutants had bigger stomata complexes. The WT and tgg mutants also showed variations for cell wall, chloroplasts, and starch grains of GCs. Abscisic acid (ABA)-treated stomata showed that the stomatal aperture was reduced in tgg1 single and tgg1 tgg2 double mutants. The data provides a basis to perform comprehensive further studies to find physiological and molecular mechanisms associated with ultrastructure differences in tgg mutants. We speculate that the absence of myrosinase alters the endogenous chemical composition, hence affecting the physical structure of plants and the plants’ physical defence barriers