Efficacy of Citrullus colocynthis seed extract on Earias vittella, Fabricius, (Lepidoptera: Noctuidae): environment sustainable approach

Abstract Earias vittellaFabricius, 1794 (Noctuidae: Lepidoptera) is deliberated to be one of the most destructive pests of cotton and okra vegetation in the world including Asia. The pest has established resistance to various synthetic insecticides. The use of bio-pesticide is one of the unconventional approaches to develop a vigorous ecosystem without harming non- target pests and beneficial natural insect fauna. In the present study, the toxicity levels of Citrullus colocynthis seed extract have been evaluated against the populations of E. vittellaunder standardized laboratory conditions. The toxic effects of C. colocynthis on development periods, protein contents and esterase activity of the life stages of E. vittella were also evaluated. The toxicity levels of methanol, ethanol, hexane, water and profenofos were evaluated on the 1st instar larvae of E. vittella. LC30 and LC80 concentrations exhibited the effectiveness of methanol-based C. colocynthis seed extract against 1st instar larvae of E. vitella. The enhanced larval and pupal periods were revealed in treated samples during the comparison with untreated samples. The intrinsic rate of increase, net reproductive rate in the LC30 and LC80 concentrations exposed larvae remained less than the control treatment. Fecundity, the esterase activity and protein contents were declined in LC30 and LC80 treated samples as compared to the control. The present findings suggest that C. colosynthis extracts based botanical insecticides are beneficial, ecosystem sustainable and can be integrated with insect management programs from environment safety perspective.

Resumo Earias vittella Fabricius, 1794 (Noctuidae: Lepidoptera) é considerada uma das pragas mais destrutivas de algodão e quiabo no mundo, incluindo a Ásia. Essa praga estabeleceu resistência a vários inseticidas sintéticos. O uso de biopesticidas é uma das abordagens não convencionais para desenvolver um ecossistema saudável sem prejudicar as pragas não alvo e a fauna natural benéfica de insetos. No presente estudo, os níveis de toxicidade do extrato de semente de Citrullus colocynthis foram avaliados nas populações de E. vittella em condições de laboratório padronizadas. Os efeitos tóxicos de C. colocynthis nos períodos de desenvolvimento, conteúdo de proteína e atividade esterase das fases de vida de E. vittella também foram avaliados. Os níveis de toxicidade de metanol, etanol, hexano, água e profenofós foram avaliados em larvas de 1º instar de E. vittella. As concentrações de LC30 e LC80 apresentaram eficácia do extrato de sementes de C. colocynthis à base de metanol contra larvas de 1º instar de E. vittella. Os períodos larval e pupal aumentados foram revelados nas amostras tratadas durante a comparação com as amostras não tratadas. A taxa intrínseca de aumento e a taxa reprodutiva líquida nas concentrações de larvas expostas LC30 e LC80 permaneceram menores do que o tratamento controle. A fecundidade, a atividade da esterase e o conteúdo de proteína diminuíram nas amostras tratadas com LC30 e LC80 em comparação com o controle. As presentes descobertas sugerem que os extratos de C. colocynthis à base de inseticidas botânicos são benéficos, sustentáveis para o ecossistema e podem ser integrados com programas de manejo de insetos do ponto de vista da segurança ambiental.

Efficacy of Citrullus colocynthis seed extract on Earias vittella, Fabricius, (Lepidoptera: Noctuidae): environment sustainable approach / Eficácia do extrato de semente de Citrullus colocynthis em Earias vittella Fabricius (Lepidoptera: Noctuidae): abordagem ambiental sustentável

Abstract Earias vittellaFabricius, 1794 (Noctuidae: Lepidoptera) is deliberated to be one of the most destructive pests of cotton and okra vegetation in the world including Asia. The pest has established resistance to various synthetic insecticides. The use of bio-pesticide is one of the unconventional approaches to develop a vigorous ecosystem without harming non- target pests and beneficial natural insect fauna. In the present study, the toxicity levels of Citrullus colocynthis seed extract have been evaluated against the populations of E. vittellaunder standardized laboratory conditions. The toxic effects of C. colocynthis on development periods, protein contents and esterase activity of the life stages of E. vittella were also evaluated. The toxicity levels of methanol, ethanol, hexane, water and profenofos were evaluated on the 1st instar larvae of E. vittella. LC30 and LC80 concentrations exhibited the effectiveness of methanol-based C. colocynthis seed extract against 1st instar larvae of E. vitella. The enhanced larval and pupal periods were revealed in treated samples during the comparison with untreated samples. The intrinsic rate of increase, net reproductive rate in the LC30 and LC80 concentrations exposed larvae remained less than the control treatment. Fecundity, the esterase activity and protein contents were declined in LC30 and LC80 treated samples as compared to the control. The present findings suggest that C. colosynthis extracts based botanical insecticides are beneficial, ecosystem sustainable and can be integrated with insect management programs from environment safety perspective.

Resumo Earias vittella Fabricius, 1794 (Noctuidae: Lepidoptera) é considerada uma das pragas mais destrutivas de algodão e quiabo no mundo, incluindo a Ásia. Essa praga estabeleceu resistência a vários inseticidas sintéticos. O uso de biopesticidas é uma das abordagens não convencionais para desenvolver um ecossistema saudável sem prejudicar as pragas não alvo e a fauna natural benéfica de insetos. No presente estudo, os níveis de toxicidade do extrato de semente de Citrullus colocynthis foram avaliados nas populações de E. vittella em condições de laboratório padronizadas. Os efeitos tóxicos de C. colocynthis nos períodos de desenvolvimento, conteúdo de proteína e atividade esterase das fases de vida de E. vittella também foram avaliados. Os níveis de toxicidade de metanol, etanol, hexano, água e profenofós foram avaliados em larvas de 1º instar de E. vittella. As concentrações de LC30 e LC80 apresentaram eficácia do extrato de sementes de C. colocynthis à base de metanol contra larvas de 1º instar de E. vittella. Os períodos larval e pupal aumentados foram revelados nas amostras tratadas durante a comparação com as amostras não tratadas. A taxa intrínseca de aumento e a taxa reprodutiva líquida nas concentrações de larvas expostas LC30 e LC80 permaneceram menores do que o tratamento controle. A fecundidade, a atividade da esterase e o conteúdo de proteína diminuíram nas amostras tratadas com LC30 e LC80 em comparação com o controle. As presentes descobertas sugerem que os extratos de C. colocynthis à base de inseticidas botânicos são benéficos, sustentáveis ​​para o ecossistema e podem ser integrados com programas de manejo de insetos do ponto de vista da segurança ambiental.

Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato.

MAIN CONCLUSION: An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor. Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts' transgene expression and protoplasts' regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that UBIQUITIN10 promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the NEOMYCIN PHOSPHOTRANSFERASE2 (NPT2) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the BABYBOOM (BBM) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.

Efficacy of Citrullus colocynthis seed extract on Earias vittella, Fabricius, (Lepidoptera: Noctuidae): environment sustainable approach.

Earias vittellaFabricius, 1794 (Noctuidae: Lepidoptera) is deliberated to be one of the most destructive pests of cotton and okra vegetation in the world including Asia. The pest has established resistance to various synthetic insecticides. The use of bio-pesticide is one of the unconventional approaches to develop a vigorous ecosystem without harming non- target pests and beneficial natural insect fauna. In the present study, the toxicity levels of Citrullus colocynthis seed extract have been evaluated against the populations of E. vittellaunder standardized laboratory conditions. The toxic effects of C. colocynthis on development periods, protein contents and esterase activity of the life stages of E. vittella were also evaluated. The toxicity levels of methanol, ethanol, hexane, water and profenofos were evaluated on the 1st instar larvae of E. vittella. LC30 and LC80 concentrations exhibited the effectiveness of methanol-based C. colocynthis seed extract against 1st instar larvae of E. vitella. The enhanced larval and pupal periods were revealed in treated samples during the comparison with untreated samples. The intrinsic rate of increase, net reproductive rate in the LC30 and LC80 concentrations exposed larvae remained less than the control treatment. Fecundity, the esterase activity and protein contents were declined in LC30 and LC80 treated samples as compared to the control. The present findings suggest that C. colosynthis extracts based botanical insecticides are beneficial, ecosystem sustainable and can be integrated with insect management programs from environment safety perspective.

Comparative transcriptome mining for terpenoid biosynthetic pathway genes in wild and cultivated species of Plantago.

Plantagos are important economical and medicinal plants that possess several bioactive secondary metabolites, such as phenolics, iridoids, triterpenes, and alkaloids. Triterpenoids are the ubiquitous and dynamic secondary metabolites that are deployed by plants for chemical interactions and protection under biotic/abiotic stress. Plantago ovata, a cultivated species, is the source of psyllium, while Plantago major, a wild species, has significant therapeutic potential. Wild species are considered more tolerant to stressful conditions in comparison to their cultivated allies. In view of this, the present study aimed to decipher the terpenoid biosynthetic pathway operative in P. ovata and P. major using a comparative transcriptomics approach. Majority of terpenoid biosynthetic genes were observed as upregulated in P. major including rate limiting genes of MVA (HMGR) and MEP (DXR) pathways and genes (α-AS, BAS, SM, and CYP716) involved in ursolic acid biosynthesis, an important triterpenoid prevalent in Plantago species. The HPLC output further confirmed the higher concentration of ursolic acid in P. major as compared to P. ovata leaf samples, respectively. In addition to terpenoid biosynthesis, KEGG annotation revealed the involvement of differentially expressed unigenes in several metabolic pathways, aminoacyl-tRNA biosynthesis, biosynthesis of antibiotics, and biosynthesis of secondary metabolites. MYB was found as the most abundant transcription factor family in Plantago transcriptome. We have been able to generate valuable information which can help in improving terpenoid production in Plantago. Additionally, the present study has laid a strong foundation for deciphering other important metabolic pathways in Plantago.

In-vitro cytotoxicity in relation to chemotypic diversity in diploid and tetraploid populations of Gentiana kurroo Royle.

ETHNOPHARMACOLOGICAL RELEVANCE: Gentiana kurroo is a multipurpose critically endangered medicinal herb prescribed as medicine in Ayurveda in India and exhibits various pharmacological properties including anti-cancer activity. The species is rich repository of pharmacologically active secondary metabolites together with secoiridoidal glycosides. AIM OF THE STUDY: The study aimed to investigate the chemical diversity in different populations/cytotypes prevailing in G. kurroo to identify elite genetic stocks in terms of optimum accumulation/biosynthesis of desired metabolites and having higher in-vitro cytotoxicity potential in relation to chemotypic diversity. MATERIAL AND METHODS: The wild plants of the species were collected from different ranges of altitudes from the Kashmir Himalayas. For cytological evaluation, the standard meiotic analysis was performed. The standard LC-MS/MS technique was employed for phytochemical analysis based on different marker compounds viz. sweroside, swertiamarin, and gentiopicroside. Different tissues such as root-stock, aerial parts, and flowers were used for chemo-profiling. Further, the methanolic extracts of diploid and tetraploid cytotypes were assessed for cytotoxic activity by using MTT assay against four different human cancer cell lines. RESULTS: The quantification of major bioactive compounds based on tissue- and location-specific comparison, as well as in-vitro cytotoxic potential among extant cytotypes, was evaluated. The comprehensive cytomorphological studies of the populations from NW Himalayas revealed the occurrence of different chromosomal races viz. n = 13, 26. The tetraploid cytotype was hitherto unreported. The tissue-specific chemo-profiling revealed relative dominance of different phytoconstituents in root-stock. There was a noticeable increase in the quantity of the analyzed compounds in relation to increasing ploidy status along the increasing altitudes. The MTT assay of methanolic extracts of diploid and tetraploid cytotypes displayed significant cytotoxicity potential in tetraploids. The root-stock extracts of tetraploids were highly active extracts with IC50 value ranges from 5.65 to 8.53 µg/mL against HCT-116 colon cancer. CONCLUSION: The chemical evaluation of major bioactive compounds in diverse cytotypes from different plant parts along different altitudes presented an appreciable variability in sweroside, swertiamarin, and gentiopicroside contents. Additionally, the concentrations of these phytoconstituents varied for cytotoxicity potential among different screened cytotypes. This quantitative difference of active bio-constituents was in correspondence with the growth inhibition percentage of different tested cancer cell lines. Thus, the present investigation strongly alludes towards a prognostic approach for the identification of elite cytotypes/chemotypes with significant pharmacological potential.

Metabolic and transcriptional analyses in response to potent inhibitors establish MEP pathway as major route for camptothecin biosynthesis in Nothapodytes nimmoniana (Graham) Mabb.

BACKGROUND: Nothapodytes nimmoniana, a plant of pivotal medicinal significance is a source of potent anticancer monoterpene indole alkaloid (MIA) camptothecin (CPT). This compound owes its potency due to topoisomerase-I inhibitory activity. However, biosynthetic and regulatory aspects of CPT biosynthesis so far remain elusive. Production of CPT is also constrained due to unavailability of suitable in vitro experimental system. Contextually, there are two routes for the biosynthesis of MIAs: the mevalonate (MVA) pathway operating in cytosol and the methylerythritol phosphate (MEP) pathway in the plastids. Determination of relative precursor flux through either of these pathways may provide a new vista for manipulating the enhanced CPT production. RESULTS: In present study, specific enzyme inhibitors of MVA (lovastatin) and MEP pathways (fosmidomycin) were used to perturb the metabolic flux in N. nimmoniana. Interaction of both these pathways was investigated at transcriptional level by using qRT-PCR and at metabolite level by evaluating secologanin, tryptamine and CPT contents. In fosmidomycin treated plants, highly significant reduction was observed in both secologanin and CPT accumulation in the range 40-57% and 64-71.5% respectively, while 4.61-7.69% increase was observed in tryptamine content as compared to control. Lovastatin treatment showed reduction in CPT (7-11%) and secologanin (7.5%) accumulation while tryptamine registered slight increase (3.84%) in comparison to control. These inhibitor mediated changes were reflected at transcriptional level via altering expression levels of deoxy-xylulose-5-phosphate reductoisomerase (DXR) and hydroxymethylglutaryl-CoA reductase (HMG). Further, mRNA expression of four more genes downstream to DXR and HMG of MEP and MVA pathways respectively were also investigated. Expression analysis also included secologanin synthase (SLS) and strictosidine synthase (STR) of seco-iridoid pathway. Present investigation also entailed development of an efficient in vitro multiplication system as a precursor to pathway flux studies. Further, a robust Agrobacterium-mediated transformed hairy root protocol was also developed for its amenability for up-scaling as a future prospect. CONCLUSIONS: Metabolic and transcriptional changes reveal differential efficacy of cytosolic and plastidial inhibitors in context to pathway flux perturbations on seco-iridoid end-product camptothecin. MEP pathway plausibly is the major precursor contributor towards CPT production. These empirical findings allude towards developing suitable biotechnological interventions for enhanced CPT production.

Jasmonate responsive transcription factor WsMYC2 regulates the biosynthesis of triterpenoid withanolides and phytosterol via key pathway genes in Withania somnifera (L.) Dunal.

KEY MESSAGE: Functional characterization of WsMYC2 via artificial microRNA mediated silencing and transient over-expression displayed significant regulatory role vis-à-vis withanolides and stigmasterol biosyntheses in Withania somnifera. Further, metabolic intensification corroborated well with higher expression levels of putative pathway genes. Additionally, copious expression of WsMYC2 in response to exogenous elicitors resulted in enhanced withanolides production. Withania somnifera, a high value multipurpose medicinal plant, is a rich reservoir of structurally diverse and biologically active triterpenoids known as withanolides. W. somnifera has been extensively pursued vis-à-vis pharmacological and chemical studies. Nonetheless, there exists fragmentary knowledge regarding the metabolic pathway and the regulatory aspects of withanolides biosynthesis. Against this backdrop, a jasmonate-responsive MYC2 transcription factor was identified and functionally characterized from W. somnifera. In planta transient over-expression of WsMYC2 showed significant enhancement of mRNA transcript levels which corroborated well with the enhanced content of withanolides and stigmasterol. Further, a comparative analysis of expression levels of some of the genes of triterpenoid pathway viz. WsCAS, WsCYP85A, WsCYP90B and WsCYP710A in corroboration with the over-expression and silencing of WsMYC2 suggested its positive influence on their regulation. These corroboratory approaches suggest that WsMYC2 has cascading effect on over-expression of multiple pathway genes leading to the increased triterpenoid biosynthesis in infiltered plants. Further, the functional validation of WsMYC2 was carried out by artificial micro-RNA mediated silencing. It resulted in significant reduction of withanolides and stigmasterol levels, indicative of crucial role of WsMYC2 in the regulation of their biosyntheses. Taken together, these non-complementary approaches provided unambiguous understanding of the regulatory role of WsMYC2 in context to withanolides and stigmasterol biosyntheses. Furthermore, the upstream promoter of WsMYC2 presented several cis-regulatory elements primarily related to phytohormone responsiveness. WsMYC2 displayed inducible nature in response to MeJA. It had substantial influence on the higher expression of WsMYC2 which was in consonance with enhanced accumulation of withanolides.

Crocetenone, a new rotenoid with an unusual trans-fused ring system from Iris crocea.

Crocetenone, a new rotenoid (1), along with five known compounds apocyanin (2), tectorigenin (3), 5,2',3'-trihydroxy-7-methoxy flavanone (4), tectoridin (5) and tectoridin glycoside (6), were isolated from the methanolic extract of the root of Iris crocea. The structure of compounds was elucidated on the basis of 1D- and 2D-NMR spectroscopic and MS analysis. Antibacterial and antioxidant activities of compounds 1-6 were evaluated. Crocetenone (1) showed a prominent antibacterial activity.

Maternal and newborn care: practices and beliefs of traditional birth attendants in Sindh, Pakistan.

Maternal mortality, infant mortality and neonatal mortality are high in Pakistan where maternal health services depend upon traditional birth attendants (TBAs). We examined the practices of TBAs in Dadu district in rural Sindh from September to November 1998 by interviewing and hosting focus group discussions with 17 TBAs. Health care personnel and other important members of the community were also interviewed. TBAs worked in areas demarcated by extended families, ethnicity or geographical access and a system of seniority was observed. Only one TBA was formally trained and antenatal and postnatal care concepts, cleanliness and equipment were inadequate. Communities trusted the TBAs and remunerated them according to factors particular to each birth. TBAs need training and to be linked with the formal health sector to effect change and to decrease maternal and neonatal mortality.

Maternal and newborn care: practices and beliefs of traditional birth attendants in Sindh, Pakistan

Maternal mortality, infant mortality and neonatal mortality are high in Pakistan where maternal health services depend upon traditional birth attendants [TBAs]. We examined the practices of TBAs in Dadu district in rural Sindh from September to November 1998 by interviewing and hosting focus group discussions with 17 TBAs. Health care personnel and other important members of the community were also interviewed. TBAs worked in areas demarcated by extended families, ethnicity or geographical access and a system of seniority was observed. Only one TBA was formally trained and antenatal and postnatal care concepts, cleanliness and equipment were inadequate. Communities trusted the TBAs and remunerated them according to factors particular to each birth. TBAs need training and to be linked with the formal health sector to effect change and to decrease maternal and neonatal mortality