Induction of systemic resistance in rice by leaf extracts of Zizyphus jujuba and Ipomoea carnea against Rhizoctonia solani

Plants accumulate a great diversity of natural products, many of which confer protective effects against phytopathogenic attack. Earlier we had demonstrated that the leaf extracts of Zizyphus jujuba and Ipomoea carnea inhibit the in vitro mycelial growth of Rhizoctonia solani, and effectively reduce the incidence of sheath blight disease in rice.⁷ Here we demonstrate that foliar application of the aqueous leaf extracts of Z. jujuba and I. carnea followed by challenge inoculation with R. solani induces systemic resistance in rice as evident from significantly increased accumulation of pathogenesis-related proteins such as chitinase, β-1,3-glucanase and peroxidase, as well as defense-related compounds such as phenylalanine ammonia-lyase and phenolic substances. Thin layer chromatographic separation of secondary metabolites revealed presence of alkaloid and terpenoid compounds in the leaf extracts of Z. jujuba that exhibited toxicity against R. solani under in vitro condition. Thus, the enhanced sheath blight resistance in rice seedlings treated with leaf extracts of Z. jujuba or I. carnea can be attributed to the direct inhibitory effects of these leaf extracts as well as their ability to elicit systemic resistance against R. solani.Key words: sheath blight, Zizyphus jujuba, Ipomoea carnea, Rhizoctonia solani, induced systemic resistance, antimicrobial compoundsSheath blight disease of rice, caused by Rhizoctonia solani, has become a major production constraint in intensive rice cropping systems where semi-dwarf, nitrogen-responsive and high-yielding rice cultivars are grown. The disease causes an annual yield loss of upto 50%.¹ R. solani is both soil- and water-borne, and can infect more than 27 families of both monocot and dicot species.² Natural host genetic resistance to R. solani has not been recorded in cultivars or wild relatives of rice.³ Several broad spectrum fungicides have been recommended for control of sheath blight, however, chemical method of disease management is neither practical due to high cost of fungicides nor sustainable as it can affect the balance of ecosystem by destroying beneficial microbial population. In addition, the environmental pollution problems associated with indiscriminate use of synthetic pesticides have prompted investigations on exploiting bio-pesticides of plant and microbial origin.Plants accumulate an enormous variety of over 100,000 secondary metabolites,⁴ which can act as pre-existing chemical inhibitors to invading pathogens and/or help strengthen defense response of host plant. The pre-formed infectional barriers in plants are generally referred to as “phytoanticipins;” whereas, the antimicrobial compounds that are synthesized de novo in response to pathogen attack are referred to as “phytoalexins.”⁵ Because of years of selective breeding leading to removal of natural products, the endogenous levels of phytoanticipins in commonly cultivated crop species are generally low and often not sufficient to fight pathogen attack, effectively.⁴ Various weed species and wild relatives of crop plants that are not subjected to selective breeding are believed to contain higher levels of antimicrobial compounds, consistent with their ability to fight invading pathogens more effectively than cultivated crop species. Identification of such weed/plant species that are enriched with antimicrobial principles, isolation of bio-active compounds from them, and application in the form of concentrated formulations to crop plants can augment their disease resistance capability by directly inhibiting the growth of pathogen and inducing defense responses. Indeed, the antimicrobial properties of tissue extracts of several weed/plant species have been reported by a number of research groups world-wide, especially in Asia and Latin America.^6–13Earlier, we had evaluated the antimicrobial activity of leaf extracts of 16 different plant species belonging to 16 different families and demonstrated that leaf extracts of most of these plant species exhibit growth-inhibitory activities against R. solani and Xanathomoas oryzae pv. oryzae (Xoo).⁷ Among these, the leaf extracts of Datura metel were found to be the most effective in inhibiting the mycelial growth and sclerotia formation of R. solani, and the growth of Xoo, as well as in reducing the incidence of sheath blight and bacterial blight diseases caused by these pathogens, respectively, under greenhouse condition.⁷ We further demonstrated that rice seedlings treated with leaf extracts of D. metel accumulated significantly higher levels of pathogenesis-related (PR) proteins and other defense related compounds following challenge inoculation with R. solani or Xoo.⁷ Our attempts to identify biologically active compounds from D. metel revealed the presence of a withanolide compound “daturilin” that exhibited remarkable antibacterial activity against Xoo.⁷Apart from D. metel, two other plants species, Zizyphus jujuba and Ipomoea carnea, were found to possess remarkable antifungal activity against R. solani.⁷ Z. jujuba is a thorny rhamnaceous plant that is widely distributed in Europe and South-eastern Asia. I. carnea of convolvulaceae family, commonly known as morning glory, is a toxic weed found in abundance in India, Brazil, the United States and other countries.¹⁴ Both of these plant species have allelopathic effect and are commonly used in folklore medicine for curing multiple diseases.^15–18 The aqueous and methanol leaf extracts of Z. jujuba and I. carnea have been found to be highly effective in reducing in vitro mycelial growth, and therefore, sclerotia production of R. solani.⁷ In the greenhouse experiments, rice seedlings sprayed with leaf extracts of Z. jujuba and I. carnea exhibited 44 and 34% reduction in severity of sheath blight disease over the control, respectively.⁷ While these findings are encouraging, the mechanisms by which the leaf extracts of Z. jujuba and I. carnea modulate defense responses in rice have not yet been explored.Plants are endowed with defense genes which remain quiescent or are expressed at basal levels in healthy plants. Activation of defense genes results in induction of systemic resistance in host plant; this defense response, designated as induced systemic resistance (ISR), plays an important role in development of disease resistance.¹⁹ The onset of ISR in plants correlates with accumulation of phytoalexins and increased activity of PR proteins such as chitinases, β-1,3-glucanases and peroxidases;^20–23 consequently, PR proteins are generally used as ISR markers.¹⁹ The classical inducers of ISR include both biotic and abiotic factors, including disease causing microorganisms themselves,^24,25 plant growth promoting rhizobacteria,^22,26 chemicals^27,28 and natural plant products.^{7,10,12,13,29,30} Plant products have been considered as one of the major groups of compounds that induce ISR. To date, extracts of at least a few plant species have been reported to contain allelopathic substances which can act as elicitors and induce systemic resistance in host plants resulting in reduction or inhibition of disease development.^7,10,12,13In the present study, with the objective of understanding the mechanisms of disease suppression by leaf extracts of Z. jujuba and I. carnea, we investigated their ability to induce ISR in rice by analyzing the activities of ISR markers including PR-proteins and other defense enzymes involved in phenylpropanoid metabolism. The changes in activities of chitinase, β-1,3-glucanase, peroxidase, phenylalanine ammonia-lyase (PAL) and phenolic compounds induced in rice seedlings that were elicited with leaf extracts (at 1:10 dilution; w/v) of Z. jujuba or I. carnea and infected with R. solani were analyzed, and compared to changes in non-elicited and uninfected seedlings. Rice seedlings that were both elicited with leaf extracts of Z. jujuba or I. carnea and infected with R. solani accumulated significantly higher levels (2–5-fold) of ISR markers as compared to non-elicited and/or uninfected seedlings (Fig. 1). About two-fold increase in activities of ISR markers was also observed in seedlings that were either infected but not elicited or elicited but not infected; however, this increase was significantly lower than the changes in seedlings that were both elicited and infected (Fig. 1). Although the activity of all ISR markers began to increase around or after 24 h post-infection, at least two distinct induction patterns were observed. For instance, the activities of chitinase and phenolic substances gradually increased to reach maximum levels at 164 h post-infection (Fig. 1A and E); whereas, the activities of β-1,3-glucanase, peroxidase and PAL reached maximum levels at 72 to 96 h post-infection and decreased thereafter (Fig. 1B–D). The leaf extracts of Z. jujuba were found slightly more effective in inducing ISR markers than the leaf extracts of I. carnea. There was no significant change in the activity of ISR markers in control seedlings sprayed with sterile distilled water (Fig. 1). Collectively, these results suggested that the leaf extracts of Z. jujuba and I. carnea have the ability to induce systemic resistance in rice seedlings infected with R. solani. The fungitoxicity of the leaf extracts of Z. jujuba and I. carnea ⁷ combined with their ability to elicit ISR is possibly responsible for low sheath blight disease incidence observed in rice seedlings treated with these leaf extracts.⁷Open in a separate windowFigure 1Activity of ISR markers and defense-related compounds in rice seedlings elicited with the leaf extracts of Zizyphus jujuba or Ipomoea carnea and challenge inoculated with Rhizoctonia solani. Total activity of chitinase (A), β-1,3-glucanase (B), peroxidase (C) phenylalanine ammonia-lyase (PAL; D) and phenolic substances (E) was analyzed in rice seedlings. The inoculation of rice seedlings with R. solani was performed 45 days after planting. Spraying of leaf extracts (1:10 dilution; w/v) of Z. jujuba or I. carnea was performed two days prior to inoculation. Tissue samples (sheath) from elicited and/or infected seedlings were collected for analysis at various time intervals.The in vitro antimicrobial and in vivo disease inhibitory effects of natural plant products are generally attributed to the allelopathic substances present in them. However, very few attempts have been made to purify and characterize active principles from bio-active natural plant products. We have previously identified a withanolide compound from leaf extracts of D. metel which exhibited antibacterial activity against Xoo.⁷ Both Z. jujuba and I. carnea are rich source of secondary metabolites including alkaloids, terpenoids, flavonoids and phenolic compounds.^31–35 To determine the composition of bio-active ingredients within the leaf extracts of Z. jujuba and I. carnea, we performed thin layer chromatographic separation of alkaloid, terpenoid and phenolic compounds. The partially purified compounds, as reported in

Blockade of tumor growth due to matrix metalloproteinase-9 inhibition is mediated by sequential activation of beta1-integrin, ERK, and NF-kappaB

We previously showed that matrix metalloproteinase (MMP)-9 inhibition using an adenovirus-mediated delivery of MMP-9 small interfering RNA (Ad-MMP-9), caused senescence in medulloblastoma cells. Regardless of whether or not Ad-MMP-9 would induce apoptosis, the possible signaling mechanism is still obscure. In this report, we demonstrate that Ad-MMP-9 induced apoptosis in DAOY cells as determined by propidium iodide and terminal deoxynucleotidyltransferase-mediated nick end labeling staining. Ad-MMP-9 infection induced the release of cytochrome c, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase. Ad-MMP-9 infection stimulated ERK, and electrophoretic mobility shift assay indicated an increase in NF-kappaB activation. ERK inhibition, using a kinase-dead mutant for ERK, ameliorated NF-kappaB activation and caspase-mediated apoptosis in Ad-MMP-9-infected cells. beta1-Integrin expression in Ad-MMP-9-infected cells also increased, and this increase was reversed by the reintroduction of MMP-9. We found that the addition of beta1 blocking antibodies inhibited Ad-MMP-9-induced ERK activation. Taken together, our results indicate that MMP-9 inhibition induces apoptosis due to altered beta1-integrin expression in medulloblastoma. In addition, ERK activation plays an active role in this process and functions upstream of NF-kappaB activation to initiate the apoptotic signal.     

Synthesis and biological evaluation of pyrazole linked benzothiazole-β-naphthol derivatives as topoisomerase I inhibitors with DNA binding ability

A series of new pyrazole linked benzothiazole-β-naphthol derivatives were designed and synthesized using a simple, efficient and ecofriendly route under catalyst-free conditions in good to excellent yields. These derivatives were evaluated for their cytotoxicity on selected human cancer cell lines. Among those, the derivatives 4j, 4k and 4l exhibited considerable cytotoxicity with IC₅₀ values ranging between 4.63 and 5.54?µM against human cervical cancer cells (HeLa). Structure activity relationship was elucidated by varying different substituents on benzothiazoles and pyrazoles. Further, flow cytometric analysis revealed that these derivatives induced cell cycle arrest in G2/M phase and spectroscopic studies such as UV–visible, fluorescence and circular dichroism studies showed that these derivatives exhibited good DNA binding affinity. Additionally, these derivatives can effectively inhibit the topoisomerase I activity. Viscosity studies and molecular docking studies demonstrated that the derivatives bind with the minor groove of the DNA.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Toxicity profiling of the ethanolic extract of Citrullus lanatus seed in rats: behavioral,biochemical and histopathological aspects

Citrullus lanatus (Cucurbitaceae) is conventionally used for the treatment of urinary tract infection, renal stones, hypertension, diabetes and diarrhoea. Current study evaluates acute and 28 days repeated toxicity ethanolic extract of C. lanatus seed (EECLS) in Wistar rats to measure its safety profile. The single dose (2000 mg/kg BW) of EECLS was administered while in 28 days repeated study 250, 500 and 1000 mg/kg BW were administered orally in rats. Parameters such as biochemical, haematological and histopathological were analysed in subacute toxicity study. During study, no apparent sign of toxicity, behavioural changes and mortality were detected in acutely exposed animals. In 28 days repeated toxicity study, rats did not show significant changes in behaviour, gross pathology, body weight, biochemical and haematological parameters. Abridged serum glucose and cholesterol levels during the study designate their roles in treatment of hyperglycaemic and hyperlipidaemic conditions. No significant difference was observed in histopathology of liver and kidneys of treated rats. The current investigation demonstrated that EECLS is non-toxic below 1000 mg/kg BW and provides protection to some body organs. The data propose that LD₅₀ of EECLS was greater than 2000 mg/kg BW and the no observed adverse effect level (NOAEL) of EECLS was at the dose of 1000 mg/kg in rats. Taken together, our finding suggests that, EECLS is safe and provides some protection to body organs; also, its extract can be used for further preclinical and clinical evaluation for its therapeutic activity.