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.
"Reduction of potassium nitrite was shown by a transparent color of the mixture. Siderophore production of isolates was tested on Chromazurol-S (CAS) agar media as described by Shin et al. (2001). Isolates were cultivated for 120 h at 28 • C. Siderophoreproducing strains formed a halo zone around the colony. "
[Show abstract][Hide abstract] 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.
"After incubation at 28 0 C for 24 h orange color indicated siderophore production and the diameter was measured. Ratio of orange halo diameter on media to colony diameter shown siderophore production as described by Sung et al., (2001). "
[Show abstract][Hide abstract] ABSTRACT: A B S T R A C T Wheat is the staple food crop all over the world. Tilletia indica causing Karnal bunt of wheat now considered alarming factor due to considerable losses in terms of crop yield. Chemical control of Karnal bunt is yet very difficult so there was need of alternate control especially biological control. Detailed survey was done for the collection of infected grains and the soil for pathogen as well as antagonistic microbe isolation. In-vitro tests were performed to check the antagonistic properties against Tilletia indica. Field evaluation of these rhizobacterial isolates with and without salicylic acid was done in BARI Bahawalpur. Already screened two susceptible advance lines and one moderately susceptible variety was selected for further studies. Seeds treated with three antagonistic rhizobacterial isolates were grown in plots already treated with salicylic acid and untreated as control. Treatments were assessed for incidence of karnal bunt in susceptible and moderately susceptible varieties of wheat. Incidence of Karnal bunt was significantly lower in antagonistic rhizobacterial treated seeds sown in salicylic acid treated plots as compared to untreated plots.
"Protease activity indicated by casein degradation was determined according to distinct zones in skim milk agar (50 ml sterilized skimmed milk mixed with 1 g agar at 55 °C) after incubation at 30 °C for 3 days. Siderophore expression was determined on CAS agar as described by Shin et al. (2001). Diversity of amplified rDNA restriction analysis For analyzing DNA fingerprint diversity of antagonistic bacteria, bacterial genomic DNA was extracted by the Mini BEST Bacterial Genomic DNA Extraction kit (TaKaRa Biotechnology Co., Ltd, Dalien, China). "
[Show abstract][Hide abstract] 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 · 1.49 Impact Factor
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