Endophytic bacterial flora in root and stem tissues of black pepper (Piper nigrum L.) genotype: isolation, identification and evaluation against Phytophthora capsici

Aim:  To isolate and identify black pepper ( Piper nigrum L) associated endophytic bacteria antagonistic to Phytophthora capsici causing foot rot disease.
Methods and Results:  Endophytic bacteria (74) were isolated, characterized and evaluated against P. capsici . Six genera belong to Pseudomonas spp (20 strains), Serratia (1 strain), Bacillus spp. (22 strains), Arthrobacter spp. (15 strains), Micrococcus spp. (7 strains), Curtobacterium sp. (1 strain) and eight unidentified strains were isolated from internal tissues of root and stem. Three isolates, IISRBP 35, IISRBP 25 and IISRBP 17 were found effective for Phytophthora suppression in multilevel screening assays which recorded over 70% disease suppression in green house trials. A species closest match (99% similarity) of IISRBP 35 was established as Pseudomonas aeruginosa ( Pseudomonas EF568931), IISRBP 25 as P. putida ( Pseudomonas EF568932), and IISRBP 17 as Bacillus megaterium ( B. megaterium EU071712) based on 16S rDNA sequencing.
Conclusion:  Black pepper associated P. aeruginosa , P. putida and B. megaterium were identified as effective antagonistic endophytes for biological control of Phytophthora foot rot in black pepper.
Significance and Impact of the Study:  This work provides the first evidence for endophytic bacterial diversity in black pepper stem and roots, with biocontrol potential against P. capsici infection.     

Free radical scavenging actions of metallothionein isoforms I and II

By employing electron spin resonance spectroscopy, we examined the free radicals scavenging effects of hepatic metallothionein (MT) isoforms I and II (MTs-I and II) on four types of free radicals. Solutions of 0.15mM of MT-I and 0.3mM of MT-II were found to scavenge the 1,1-diphenyl-2-picrylhydrazyl radicals (1.30 × 10¹⁵ spins/ml) completely. In addition, both isoforms exhibited total scavenging action against the hydroxyl radicals (1.75 × 10¹⁵ spins/ml) generated in a Fenton reaction. Similarly, 0.3mM of MT-I scavenged almost 90% of the superoxide (2.22 × 10¹⁵ spins/ml) generated by the hypoxanthine and xanthine oxidase system, while a 0.3mM MT-II solution could only scavenge 40% of it. By using 2,2,6,6-tetramethyl-4-piperidone as a “spin-trap” for the reactive oxygen species (containing singlet oxygen, superoxide and hydroxyl radicals) generated by photosensitized oxidation of riboflavin and measuring the relative signal intensities of the resulting stable nitroxide adduct, 2,2,6,6-tetramethyl-4-piperidine-1-oxyl, we observed that MT-II (0.3 mM) could scavenge 92%, while MT-I at 0.15 mM μl/ml concentrations could completely scavenge all the reactive species (2.15 × 10¹⁵ spins/ml) generated.

The results of these studies suggest that although both isoforms of MT are able to scavenge free radicals, the MT-I appears to be a superior scavenger of superoxide and 1,1 diphenyl-2-picrylhydrazyl radicals.     

Localization of SpoVAD to the inner membrane of spores of Bacillus subtilis

The products of the hexacistronic spoVA operon of Bacillus subtilis may be involved in the transport of dipicolinic acid into the forespore during sporulation and its release during spore germination. The major hydrophilic coding region of B. subtilis spoVAD was cloned, the protein was expressed in Escherichia coli as a His tag fusion protein, and a rabbit antiserum was raised against the purified protein. Western blot analyses of fractions from B. subtilis spores showed that SpoVAD is an integral inner membrane protein present at levels >50-fold higher than those of the spore's nutrient germinant receptors that are also present in the inner membrane. SpoVAD also persisted in outgrowing spores.     

Whole-genome sequence analysis of Pseudomonas syringae pv. phaseolicola 1448A reveals divergence among pathovars in genes involved in virulence and transposition

Pseudomonas syringae pv. phaseolicola, a gram-negative bacterial plant pathogen, is the causal agent of halo blight of bean. In this study, we report on the genome sequence of P. syringae pv. phaseolicola isolate 1448A, which encodes 5,353 open reading frames (ORFs) on one circular chromosome (5,928,787 bp) and two plasmids (131,950 bp and 51,711 bp). Comparative analyses with a phylogenetically divergent pathovar, P. syringae pv. tomato DC3000, revealed a strong degree of conservation at the gene and genome levels. In total, 4,133 ORFs were identified as putative orthologs in these two pathovars using a reciprocal best-hit method, with 3,941 ORFs present in conserved, syntenic blocks. Although these two pathovars are highly similar at the physiological level, they have distinct host ranges; 1448A causes disease in beans, and DC3000 is pathogenic on tomato and Arabidopsis. Examination of the complement of ORFs encoding virulence, fitness, and survival factors revealed a substantial, but not complete, overlap between these two pathovars. Another distinguishing feature between the two pathovars is their distinctive sets of transposable elements. With access to a fifth complete pseudomonad genome sequence, we were able to identify 3,567 ORFs that likely comprise the core Pseudomonas genome and 365 ORFs that are P. syringae specific.     

Structural features of the sulphated polysaccharide from a green seaweed, Spongomorpha indica.

A sulphated heteropolysaccharide, [alpha]D +59 degrees, was isolated from a green seaweed, Spongomorpha indica, by extraction with ammonium oxalate. The polymer is composed of arabinose, xylose, galactose and glucose in the ratio 8.9:1.0:12.0:1.0. Studies showed that the polysaccharide is a complex and multilinked polymer containing arabinose in both furanose and pyranose forms. The core of the polysaccharide is composed of 1,4-linked galactose units. The arabinofuranose units are present as non-reducing end units, as well as jointed through 1,3- and 1,2-linkages. The majority of the arabinopyranose units are joined through 1,4-linkages. Xylose is present as a branch terminating unit. Glucose is joined through 1,4-linkages. Both arabinose and galactose carry branches. Sulphate groups are present on some of the arabinose units at C-2 and on some of the galactose units at C-2 and C-3.     

A statistical analysis of the effects of urease pre-treatment on the measurement of the urinary metabolome by gas chromatography–mass spectrometry

Urease pre-treatment of urine has been utilized since the early 1960s to remove high levels of urea from samples prior to further processing and analysis by gas chromatography–mass spectrometry (GC–MS). Aside from the obvious depletion or elimination of urea, the effect, if any, of urease pre-treatment on the urinary metabolome has not been studied in detail. Here, we report the results of three separate but related experiments that were designed to assess possible indirect effects of urease pre-treatment on the urinary metabolome as measured by GC–MS. In total, 235 GC–MS analyses were performed and over 106 identified and 200 unidentified metabolites were quantified across the three experiments. The results showed that data from urease pre-treated samples (1) had the same or lower coefficients of variance among reproducibly detected metabolites, (2) more accurately reflected quantitative differences and the expected ratios among different urine volumes, and (3) increased the number of metabolite identifications. Overall, we observed no negative consequences of urease pre-treatment. In contrast, urease pre-treatment enhanced the ability to distinguish between volume-based and biological sample types compared to no treatment. Taken together, these results show that urease pre-treatment of urine offers multiple beneficial effects that outweigh any artifacts that may be introduced to the data in urinary metabolomics analyses.     

Inductively coupled plasma-optical emission spectrometry for on-line determination of multi trace elements in environmental samples after preconcentration by using Borassus flabellifer inflorescence loaded with 2-propylpiperidine-1-carbodithioate

A procedure was developed for the determination of Cu, Mn, Pb, Cd, Co, Cr, and Zn in water samples by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES) after preconcentration on synthesized 2-propylpiperidine-1-carbodithioate supported by Borassus flabellifer Inflorescence (BFI). The sorbed element was subsequently eluted with 0.4 M HNO3, and the acid eluates were analyzed by ICP-OES. The influence of various parameters such as pH, flow rate of sample, eluent concentration, volume of the sample, and volume of eluent were investigated. Under the optimal conditions, Cu, Mn, Pb, Cd, Co, Cr, and Zn in aqueous samples were concentrated ca. 100-fold. Recoveries were obtained by the proposed method in the range of 97.8-99.9%. This method was also applied for the analysis of spiked, natural waters and soil samples. The results provide strong evidence to support the hypothesis of an adsorption mechanism.     

Light-dependent transformation of anthranilate to indole by Rhodobacter sphaeroides OU5

Rhodobacter sphaeroides OU5 transformed anthranilate (2 mM) to an indole (0.7 mM) in a light-dependent process. Photobiotransformation was enhanced by tricarboxylic acid cycle intermediates and the indole formed was identified as 2,3 dihydroxy indole. Journal of Industrial Microbiology & Biotechnology (2000) 24, 219–221. Received 16 September 1999/ Accepted in revised form 20 December 1999