Tehmina Anjum

Identification of a Potential ISR Determinant from Pseudomonas aeruginosa PM12 against Fusarium Wilt in Tomato

Biocontrol of plant diseases through induction of systemic resistance is an environmental friendly substitute to chemicals in crop protection measures. Different biotic and abiotic elicitors can trigger the plant for induced resistance. Present study was designed to explore the potential of Pseudomonas aeruginosa PM12 in inducing systemic resistance in tomato against Fusarium wilt. Initially the bioactive compound, responsible for ISR, was separated and identified from extracellular filtrate of P. aeruginosa PM12. After that purification and characterization of the bacterial crude extracts was carried out through a series of organic solvents. The fractions exhibiting ISR activity were further divided into sub-fractions through column chromatography. Sub fraction showing maximum ISR activity was subjected to Gas chromatography/mass spectrometry for the identification of compounds. Analytical result showed three compounds in the ISR active sub-fraction viz: 3-hydroxy-5-methoxy benzene methanol (HMB), eugenol and tyrosine. Subsequent bioassays proved that HMB is the potential ISR determinant that significantly ameliorated Fusarium wilt of tomato when applied as soil drench method at the rate of 10 mM. In the next step of this study, GC-MS analysis was performed to detect changes induced in primary and secondary metabolites of tomato plants by the ISR determinant. Plants were treated with HMB and Fusarium oxysporum in different combinations showing intensive re- modulations in defense related pathways. This work concludes that HMB is the potential elicitor involved in dynamic reprogramming of plant pathways which functionally contributes in defense responses. Furthermore the use of biocontrol agents as natural enemies of soil borne pathogens besides enhancing production potential of crop can provide a complementary tactic for sustainable integrated pest management.

Screening of Sunflower Varieties for Their Herbicidal Potential Against Common Weeds of Wheat

ABSTRACT Sunflower is a putative source of bioactive allelochemicals, especially phenolics and terpenoids. Because qualitative and quantitative variation is well documented in sunflower types, we screened three commercially grown varieties of sunflower (Hybrid Supper-25, Gulshan-98 and Hybrid Suncross-42) through aqueous extract bioassays against five selected weeds of wheat (i.e., Chenopodium album, Rumex dentatus, Coronopus didymus, Phalaris minor, and Medicago polymorpha). Sunflower proved to be a potential source of allelochemicals as natural herbicides for weed management of selected weeds. The allelopathic effect was found to be dosage dependant, as it gradually increased as the concentration use from 30% to 50%. The lower concentrations of 10% and 20% showed stimulatory effects or insignificant weed reduction. C. album and R. dentatus were found to be the most sensitive to various treatments. The studies were extended to pot trials to determine the ecological significance of the laboratory findings.

Phenylacetic Acid Is ISR Determinant Produced by Bacillus fortis IAGS162, Which Involves Extensive Re-modulation in Metabolomics of Tomato to Protect against Fusarium Wilt

Bacillus fortis IAGS162 has been previously shown to induce systemic resistance in tomato plants against Fusarium wilt disease. In the first phase of current study, the ISR determinant was isolated from extracellular metabolites of this bacterium. ISR bioassays combined with solvent extraction, column chromatography and GC/MS analysis proved that phenylacetic acid (PAA) was the potential ISR determinant that significantly ameliorated Fusarium wilt disease of tomato at concentrations of 0.1 and 1 mM. In the second phase, the biochemical basis of the induced systemic resistance (ISR) under influence of PAA was elucidated by performing non-targeted whole metabolomics through GC/MS analysis. Tomato plants were treated with PAA and fungal pathogen in various combinations. Exposure to PAA and subsequent pathogen challenge extensively re-modulated tomato metabolic networks along with defense related pathways. In addition, various phenylpropanoid precursors were significantly up-regulated in treatments receiving PAA. This work suggests that ISR elicitor released from B. fortis IAGS162 contributes to resistance against fungal pathogens through dynamic reprogramming of plant pathways that are functionally correlated with defense responses.

Mass spectrometric identification and toxicity assessment of degraded products of aflatoxin B1 and B2 by Corymbia citriodora aqueous extracts.

This study explores the detoxification potential of Corymbia citriodora plant extracts against aflatoxin B1 and B2 (AFB1; 100 μg L−1 and AFB2; 50 μg L−1) in In vitro and In vivo assays. Detoxification was qualitatively and quantitatively analyzed by TLC and HPLC, respectively. The study was carried out by using different parameters of optimal temperature, pH and incubation time period. Results indicated that C. citriodora leaf extract(s) more effectively degrade AFB1 and AFB2 i.e. 95.21% and 92.95% respectively than C. citriodora branch extract, under optimized conditions. The structural elucidation of degraded toxin products was done by LCMS/MS analysis. Ten degraded products of AFB1 and AFB2 and their fragmentation pathways were proposed based on molecular formulas and MS/MS spectra. Toxicity of these degraded products was significantly reduced as compared to that of parent compounds because of the removal of double bond in the terminal furan ring. The biological toxicity of degraded toxin was further analyzed by brine shrimps bioassay, which showed that only 17.5% mortality in larvae was recorded as compared to untreated toxin where 92.5% mortality was observed after 96hr of incubation. Therefore, our finding suggests that C. citriodora leaf extract can be used as an effective tool for the detoxification of aflatoxins.

Sunflower phytochemicals adversely affect wheat yield.

Various researchers have reported the weedicidal potential of sunflower when used as incorporation, mulch or aqueous extract without noticing its effect on crop. This study was planned to investigate the harmful effects of sunflower phytochemicals on wheat varieties. Early laboratory experiments were performed on four wheat varieties, i.e. Inqlab-91, Punjab-96, Pasban-90 and Uqab-2000. The aqueous extract of three sunflower varieties tested against wheat varieties significantly decreased biomass of wheat seedlings, especially at concentrations of 40% and 50%. However, wheat variety Punjab-96 resisted the most, which was selected for further trials. This detrimental effect was also noticed in pot trials, the extent of which varied with the age of the wheat seedlings and number of sprays. Three early sprays with one week intervals showed maximum losses to the crop plant. The study suggests the use of sunflower extracts for the management of weeds that emerge 3–4 weeks after wheat seedlings. However, the study discourages the use of sunflower mulch or incorporation in wheat fields for weed management.

Hypersensitive response - A biophysical phenomenon of producers.

Hypersensitive response/reaction is a form of the cellular demise frequently linked alongside plant resistance against pathogen infection. Main transducers for this reaction are the intermediates of reactive oxygen and ion fluxes which are plausibly needed for hypersensitive response (Hpr Sen Rsp). An immediate and enormous energy production and its intra-cellular biochemical conduction are imperative for an Hpr Sen Rsp to be occurred. A number of studies proved that there are such diverse types of factors involved in triggering of Hpr Sen Rsp that morphologies of dead cells have become a vast topic of study. Hpr Sen Rsp could play a frolic role in plants as certain programmed cellular disintegrations in other organisms, to restrict pathogen growth. In fact, Hpr Sen Rsp can be involved in all types of tissues and most of the developmental stages.

Isolation of bioactive allelochemicals from sunflower (variety Suncross-42) through fractionation-guided bioassays.

Plants are rich source of biologically active allelochemicals. However, natural product discovery is not an easy task. Many problems encountered during this laborious practice can be overcome through the modification of preliminary trials. Bioassay-directed isolation of active plant compounds can increase efficiency by eliminating many of the problems encountered. This strategy avoids unnecessary compounds, concentrating on potential components and thus reducing the cost and time required. In this study, a crude aqueous extract of sunflower leaves was fractionated through high performance liquid chromatography. The isolated fractions were checked against Chenopodium album and Rumex dentatus. The fraction found active against two selected weeds was re-fractionated, and the active components were checked for their composition. Thin layer chromatography isolated a range of phenolics, whereas the presence of bioactive terpenoids was confirmed through mass spectroscopy and nuclear magnetic resonance spectroscopy.

Structural Elucidation and Toxicity Assessment of Degraded Products of Aflatoxin B1 and B2 by Aqueous Extracts of Trachyspermum ammi

In this study aqueous extract of seeds and leaves of Trachyspermum ammi were evaluated for their ability to detoxify aflatoxin B1 and B2 (AFB1; 100 μg L−1 and AFB2; 50 μg L−1) by in vitro and in vivo assays. Results indicated that T. ammi seeds extract was found to be significant (P < 0.05) in degrading AFB1 and AFB2 i.e., 92.8 and 91.9% respectively. However, T. ammi leaves extract proved to be less efficient in degrading these aflatoxins, under optimized conditions i.e., pH 8, temperature 30°C and incubation period of 72 h. The structural elucidation of degraded toxin products by LCMS/MS analysis showed that eight degraded products of AFB1 and AFB2 were formed. MS/MS spectra showed that most of the products were formed by the removal of double bond in the terminal furan ring and modification of lactone group indicating less toxicity as compared to parent compounds. Brine shrimps bioassay further confirmed the low toxicity of degraded products, showing that T. ammi seeds extract can be used as an effective tool for the detoxification of aflatoxins.

Production of Cyclosporine A by Submerged Fermentation from a Local Isolate of Penicillium fellutanum

Six locally isolated strains of Penicillium were checked for their potential to produce cyclosporine A through submerged fermentation. The medium used for drug production was composed of glucose, 5%; peptone, 1%; KH2PO4, 0.5% and KCl, 0.25% (w/v). Butyl acetate was used to extract the fermentation medium for cyclosporine ′A′ analysis. The confirmation analysis was done through high performance liquid chromatography and the chromatograms obtained were compared with that of Sandimmun Neoral ®capsule (Novartis) containing 100 mg of cyclosporine and with the external standard cyclosporine A 98.5% pure. Only chromatogram of Penicillium fellutanum (FCBP 937) isolated from Guava fruit showed a peak at 2.768, which was comparable with both the standards. The amount of drug calculated was 16.18 μg/ml.

First Report of Curvularia lunata Causing Leaf Spots on Sorghum bicolor from Pakistan

In October 2012, reddish brown, oblong lesions with chlorotic centers were observed on the leaves of Sorghum bicolor in Punjab Province, Pakistan. Early symptoms appeared as reddish brown circular spots on the leaves. These spots increased in size and coalesced to form oblong lesions. Entire fields were severely affected by the disease. Pathogen isolations were made on malt extract agar (MEA) media. Symptomatic leaf samples were cut into 4 to 6 mm2 pieces, surface sterilized (10% bleach for 1 min, 90% ethanol for 30 sec) and rinsed in sterilized water several times, followed by air drying. These samples were plated onto 2% MEA media, supplemented with 10 mg/liter chloramphenicol, and incubated at 25°C for 6 days in the dark. A mitosporic fungus of dark brown colony, bearing large stroma, appeared on the media. Conidiophores were brown, septate, geniculate, simple or unbranched, with dark brown scar. Conidia were brown, straight to pyriform, with 3 to 4 cells, with large and curved central cells, smooth walled, ranging in size from 7.3 to 21.26 μm, and produced apically in a sympodial manner. Based on morphological characteristics, the pathogen was identified as Curvularia lunata (Wakk.) Boedijn. (1,2). Morphological identification was also confirmed by the First Fungal Culture Bank of Pakistan (FCBP), Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan, and samples were submitted to FCBP (Accession No. 1201). The fungus was further identified by amplifying internal transcribed spacer region sequences (ITS1, rDNA, ITS2) by using ITS4 and ITS5 primers (4). The resulting 584-bp sequence was submitted to GenBank with Accession No. HG326308. This sequence showed 99% homology with C. lunata strain pingxiang (GenBank Accession No. JQ701897), causing leaf spots of lotus in China. Pathogenicity assay was conducted on 20-day-old seedlings of S. bicolor variety Indian Gold, grown from surface sterilized seeds. Fifteen replicate plants were sprayed with a spore suspension of 1 × 106 spore/ml in distilled sterilized water, prepared from 1-week-old fungal culture, grown in the dark on 2% MEA media. Five replicate plants were sprayed with distilled sterilized water as control. Plants were covered with transparent polyethylene bags to retain moisture and enhance disease development, and kept in a greenhouse at ~30°C. Bags were removed after 5 days of incubation. Inoculated plants developed lesions similar to those observed on naturally infected plants. No symptoms were observed on control plants. The pathogen was re-isolated from infected leaves, and the morphology features were again studied, matching those of the pathogen isolated from field samples. Curvularia leaf spot diseases, caused by different Curvularia species, have been previously found on many grass species worldwide (3). To our knowledge, this is the first report of C. lunata leaf spots on S. bicolor in Pakistan. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Kew, Surrey, England, 1971. (2) F. B. Rocha et al. Austral. Plant Pathol. 33:601, 2004. (3) J. D. Smith et al. Fungal diseases of amenity turf grasses. E & F.N. Spon., New York, 1989. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.