CAS agar diffusion assay for the measurement of siderophores in biological fluids
ABSTRACT We developed a simple and universal method, by modifying the universal CAS (Chrome azurol S) assay, measuring siderophores in various biological fluids. We named the assay as CAS agar diffusion (CASAD) assay. CAS plate devoid of nutrients was prepared by using Bacto-agar (1.5%, w/v) as a matrix. Holes with 5-mm-diameter were punched on the CAS agar plate. Each hole was added by 35 microl of the test fluids containing Desferal that was twofold serially diluted. After incubating at 37 degrees C or room temperature for 4-8 h, the size of orange haloes formed around the holes was measured. The size of orange haloes correlated well with the concentration of Desferal in all the biological fluids tested in this study. CASAD assay showed consistent results in wide pH range from 5 to 9. Addition of iron to the test fluids containing Desferal decreased the size of orange haloes in a dose-dependent manner, which suggests that the CASAD assay detects only iron non-bound siderophore. These results suggest that CASAD assay would serve as a simple, stable, and highly reproducible test for screening and quantitative siderophore analysis in biological fluids.
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ABSTRACT: Selenium (Se)-rich plants may be used to provide dietary Se to humans and livestock, and also to clean up Se-polluted soils or waters. This study focused on endophytic bacteria of plants that hyperaccumulate selenium (Se) to 0.5–1% of dry weight. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to compare the diversity of endophytic bacteria of hyperaccumulators Stanleya pinnata (Brassicaceae) and Astragalus bisulcatus (Fabaceae) with those from related non-accumulators Physaria bellii (Brassicaceae) and Medicago sativa (Fabaceae) collected on the same, seleniferous site. Hyperaccumulators and non-accumulators showed equal T-RF diversity. Parsimony analysis showed that T-RFs from individuals of the same species were more similar to each other than to those from other species, regardless of plant Se content or spatial proximity. Cultivable endophytes from hyperaccumulators S. pinnata and A. bisulcatus were further identified and characterized. The 66 bacterial morphotypes were shown by MS MALDI-TOF Biotyper analysis and 16S rRNA gene sequencing to include strains of Bacillus, Pseudomonas, Pantoea, Staphylococcus, Paenibacillus, Advenella, Arthrobacter, and Variovorax. Most isolates were highly resistant to selenate and selenite (up to 200 mM) and all could reduce selenite to red elemental Se, reduce nitrite and produce siderophores. Seven isolates were selected for plant inoculation and found to have plant growth promoting properties, both in pure culture and when co-cultivated with crop species Brassica juncea (Brassicaceae) or M. sativa. There were no effects on plant Se accumulation. We conclude that Se hyperaccumulators harbor an endophytic bacterial community in their natural seleniferous habitat that is equally diverse to that of comparable non-accumulators. The hyperaccumulator endophytes are characterized by high Se resistance, capacity to produce elemental Se and plant growth promoting properties.Frontiers in Plant Science 03/2015; 6:1-17. DOI:10.3389/fpls.2015.00113 · 3.64 Impact Factor
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ABSTRACT: A total of 1,487 bacterial isolates were obtained from the rhizosphere, phyllosphere, endorhiza and endosphere of field-grown pepper. In a dual assay, 232 isolates displayed the antagonistic activity towards Phytophthora capsici L.; 36.6 % and 39.2 % of them were obtained from the rhizosphere and phyllosphere, respectively. 40 of the 232 antagonistic isolates producing inhibition zones of at least 5 mm in diameter were assessed for production of siderophores and chitinase, cellulose, and protease activity. These 40 isolates fell into 15 groups according to 90 % similarity of the banding patterns obtained by amplified ribosomal DNA restriction analysis (ARDRA). Seventeen isolates spanning the 15 groups were evaluated in greenhouse tests for their ability to control Phytophthora blight of pepper. Biocontrol efficacy ranged from 0.7 % to 92.3 %, with three isolates (B1301, R98, and PX35) exhibiting maximum ability to reduce the disease severity (83.5 %, 92.3 % and 83.5 %, respectively). Based on 16S rDNA sequencing, these isolates were identified as Bacillus cereus (B1301), Chryseobacterium sp (R98) and Bacillus cereus (PX35). This is the first report that Chryseobacterium sp. (R98) can function as a biocontrol agent of Phytophthora blight.European Journal of Plant Pathology 07/2012; DOI:10.1007/s10658-012-0057-7 · 1.71 Impact Factor
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ABSTRACT: Tomato (Lycopersicon esculentum) is one of the major vegetable crops, grown all over the country under diverse climatic conditions. In India, estimated area of tomato is 634 thousand hectares, production is about 12433 thousand MT and productivity 19.6 MT/ hectare (Anonymous, 2010). Among various fungal, bacterial, and viral diseases in tomato, bacterial wilt caused by Ralstonia solanacearum race 1 biovar 3 is one of the most important diseases in India and mostly confined to coastal and hilly areas including Goa, Karnataka, Kerala, Maharastra, Orissa, Jharkhand, West Bengal and hilly states, like Himachal Pradesh, Jammu & Kashmir and Uttarakhand (Devi and Menon, 1980; Singh et al., 2010). This disease causes severe loss varying from 10-90% in different climates in India (Mishra et al., 1995). Management of bacterial wilt of tomato is difficult due to soil borne nature of the pathogen. Chemical treatment of soil is one of the options for management but bears risk of soil and water pollution. Pesticide residues have detrimental effects on human, plant and soil health and leads to development of mutant resistant to pesticides (Gerhardson, 2002). Hence, a biocontrol measure employing antagonistic bacterial agents is an attractive option (Han et al., 2005). Biological control of bacterial diseases of solanaceous crops caused by R. solanacearum through the antagonistic bacteria has been reported earlier (Shekhawat et al., 1992; Myint and Ranamukhaarachchi, 2006). Among various bacterial antagonists, the endospore forming gram positive Bacillus subtilis is reported to be a good biocontrol agent and plant growth promoting bacterium (Soohee Chung et al., 2008; Myint and Ranamukhaarachchi, 2006), which rapidly colonizes the rhizosphere and suppresses deleterious microorganisms as well as soil borne pathogens (Myint and Ranamukhaarachchi, 2006). Besides antagonistic ability, Bacillus spp. can either directly or indirectly facilitate rooting (Mayak et al., 1999) and growth of plants (Glick, 1995) possibly due to production of plant growth promoting substances like indole acetic acid, phosphate solubilization, ammonia, and siderophore (Nautiyal, 1999; Mathurot et al., 2009). Classical identification methods based on morphological, biochemical, physiological parameters are too laborious and hence not applicable for the purpose of a rapid screening of bacteria. Therefore, rapid and precise DNA based techniques are valuable to distinguish bacteria at species and subsp. level. In this context, a molecular marker based on 16S rRNA sequence was developed to differentiate Bacillus species (Wattiau et al., 2001).