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Journal: Letters in applied microbiology

145

Plant-growth promoting rhizobacteria (PGPR) are studied as complements/alternatives to chemical fertilization in agriculture. Poor information however, exists on the potential of PGPR from undisturbed ecosystems. Here, we evaluated the plant growth-promoting (PGP) effect of rhizobacterial consortia from undisturbed Chilean arid-ecosystems (Consortium C1) and agro-ecosystems (Consortium C2) on plant biomass production. The PGP effects of C1 and C2 were assayed in wheat seedlings (Triticum aestivum L.) grown in pots under growth chamber conditions and in pots placed in an open greenhouse under natural conditions, using two different Chilean Andisols (Piedras Negras and Freire series) kept either at 30% or 60% of their maximum water holding capacity (MWHC). PGP effects depended on the soil type, MWHC and the growth conditions tested. Although both consortia showed PGB effects in artificial soils relative to controls in growth chambers, only C1 provoked a PGP effect at 60% MWHC in phosphorus-poor soil of the ‘Piedras Negras’ series. At natural conditions, however, only C1 exhibited statistically significant PGP effects at 30% MWHC in ‘Piedras Negras’, yet and most importantly allowed to maintain similar plant biomass as at 60% MWHC. Our results support possible applications of rhizobacterial consortia from arid ecosystems to improve wheat growth in Chilean Andisols under water shortage conditions. This article is protected by copyright. All rights reserved.

Concepts: Statistical significance, Soil, Wheat, Common wheat, Spelt, All rights reserved, Copyright, Biomass

31

The current outbreak of a novel SARS-like coronavirus, 2019_nCoV, illustrated difficulties in identifying a novel coronavirus and its natural host, as the coding sequences of various Betacoronavirus species can be highly diverse. By means of whole-genome sequence comparisons, we demonstrate that the non-coding flanks of the viral genome can be used to correctly separate the recognized four betacoronavirus subspecies. The conservation would be sufficient to define target sequences that could, in theory, classify novel virus species into their subspecies. Only 253 upstream non-coding sequences of Sarbecovirus are sufficient to identify genetic similarities between species of this subgenus. Further, it was investigated which bat species have commercial value in China, and would thus likely be handled for trading purposes. A number of coronavirus genomes have been published that were obtained from such bat species. These bats are used in Traditional Chinese Medicine, and their handling poses a potential risk to cause zoonotic coronavirus epidemics.

28

A simple method was developed for isolating DNA from jute seed, which contains high amounts of mucilage and secondary metabolites, and a PCR protocol was standardized for detecting the seedborne pathogen Macrophomina phaseolina. The cetyl trimethyl ammonium bromide method was modified with increased salt concentration and a simple sodium acetate treatment to extract genomic as well as fungal DNA directly from infected jute seed. The Miniprep was evaluated along with five other methods of DNA isolation in terms of yield and quality of DNA and number of PCR positive samples. The Miniprep consistently recovered high amounts of DNA with good spectral qualities at A260/A280. The DNA isolated from jute seed was found suitable for PCR amplification. Macrophomina phaseolina could be detected by PCR from artificially inoculated as well as naturally infected jute seeds. The limit of PCR-based detection of M. phaseolina in jute seed was determined to be 0·62 × 10(-7)  CFU g(-1) seed. SIGNIFICANCE AND IMPACT OF THE STUDY: Stem rot caused by Macrophomina phaseolina is the most important disease of jute, a bast fibre crop. Seedborne infection of the pathogen is generally detected by conventional methods such as blotter method and agar-plate method followed by microscopy. But, these techniques are time-consuming and not sensitive. In the present investigation, M. phaseolina was detected from jute seeds by PCR, which is a rapid and reliable technique. However, high contents of mucilage and secondary metabolites in jute seed hinder DNA isolation and PCR amplification. To address these problems, we developed a Miniprep which yielded a sufficient amount of good quality DNA as compared to other methods and standardized a PCR protocol, which could amplify the fungal DNA present in seed. It would enable efficient PCR-based detection of M. phaseolina from large number of jute seed lots.

Concepts: DNA, DNA sequencing, Hemp, Jute, Corchorus olitorius, Corchorus, Bast fibre, Kenaf

28

Naegleria spp. is a free-living amoeba that can be found in the natural environment. A number of Naegleria spp. can cause fatal infections in the central nervous system in humans and animals, and the most important source of infection is through direct water contact. In this study, water samples from various thermal springs were taken from four thermal spring areas. Naegleria spp. was detected via culture confirmation and molecular taxonomic identification. Among the 60 samples obtained, Naegleria spp. was identified in 26 (43·3%) samples. The identified species included Naegleria australiensis, Naegleria gruberi, Naegleria lovaniensis and Naegleria mexicana. The presence of living Naegleria spp. was significantly associated with elevated pH value in the water sample. SIGNIFICANCE AND IMPACT OF STUDY: In this study, we examined the presence of living Naegleria spp. in thermal spring waters in south-eastern Taiwan. Naegleria spp. was isolated and culture-confirmed from thermal spring water. Naegleria fowleri was not found in all water samples, and Naegleria australiensis was the most common Naegleria genotype.

Concepts: Central nervous system, Nervous system, Water, Spring, Hot spring, Geyser, Naegleria fowleri, Naegleria

28

The aim of this study was to develop novel anaerobic media using gellan gum for the isolation of previously uncultured rumen bacteria. Four anaerobic media, a basal liquid medium (BM) with agar (A-BM), a modified BM (MBM) with agar (A-MBM), an MBM with phytagel (P-MBM) and an MBM with gelrite (G-MBM) were used for the isolation of rumen bacteria and evaluated for the growth of previously uncultured rumen bacteria. Of the 214 isolates composed of 144 OTUs, 103 isolates (83 OTUs) were previously uncultured rumen bacteria. Most of the previously uncultured strains were obtained from A-MBM, G-MBM and P-MBM, but the predominant cultural members, isolated from each medium, differed. A-MBM and G-MBM showed significantly higher numbers of different OTUs derived from isolates than A-BM (P < 0·05). The Shannon index indicated that the isolates of A-MBM showed the highest diversity (H' = 3·89) compared with those of G-MBM, P-MBM and A-BM (H' = 3·59, 3·23 and 3·39, respectively). Although previously uncultured rumen bacteria were isolated from all media used, the ratio of previously uncultured bacteria to total isolates was increased in A-MBM, P-MBM and G-MBM.

Concepts: Ratio, Agar, E number, Diversity index, Shannon index, Gellan gum, Sphingomonas

28

In recent years, functional fabrics possessing antimicrobial activity have drawn significant interest because antibiotic resistance is becoming widespread among pathogenic microorganisms. The aim of this study was to produce microcapsules incorporating ozonated red pepper seed oil with antimicrobial properties and apply them to nonwoven fabrics to prepare functional textiles. Red pepper seed oil (RPSO) was ozonated and microencapsulated via a complex coacervation method using gelatin (GE) and gum arabic (GA) as wall materials. While microencapsulation yield and oil loading decreased with increases in the amount of surfactant, the mean particle size increased. The antimicrobial activity of the oil was tested via the disc diffusion method. The microcapsules were also tested using the agar well method. While RPSO had no effect on the test microorganisms, the ozonated red pepper seed oil (ORPSO) and microcapsules containing ORPSO were found to be active against the test microorganisms. The microcapsules were then applied to nonwoven fabric using the padding method to produce a disposable functional textile. The microcapsule-impregnated functional fabrics provided a 5log decrease in 1 h. It is therefore possible to functionalize nonwoven fabrics to have antimicrobial activity against antibiotic resistant microorganisms, using microcapsules containing ozonated red pepper seed oil. © 2012 The Authors Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Concepts: Bacteria, Microbiology, Antibiotic resistance, Cotton, Microorganism, Textile, Fabrics, Nonwoven Fabric

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Contamination of food industrial environments and recontamination of finished products by Chrysonilia sitophila and Hyphopichia burtonii has long been a serious problem for the bakery industries. As one of the most common ways to slow down or avoid fungal spoilage on bakery products is the use of ethanol, in the present work the effect of this substance has been assessed on growth of two of the most frequently occurring associated moulds, C. sitophila and H. burtonii, by means of tests on both synthetic media and sliced bread. Test on synthetic media: H. burtonii was less markedly affected in lag-phase duration and radial growth rates by the addition of ethanol to DG18 and the reduction of incubation temperature than C. sitophila that failed to grow at the highest concentrations of ethanol tested (2.0% and 4.0% at 15°C; 4.0% at 25°C). Test on sliced bread: ethanol proved to be effective to prevent spoilage by C. sitophila even at the lowest concentration tested (0.8%, wt/wt), while higher concentrations (2.0%, wt/wt) were needed to prevent spoilage by H. burtonii. This article is protected by copyright. All rights reserved.

Concepts: Effect, Yeast, Concentration, All rights reserved, Bread, Copyright, Mold, Sliced bread

27

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a promising tool to rapidly characterize Staphylococcus aureus. Different protocols have been employed, but effects of experimental factors, such as culture condition and sample preparation, on spectrum quality and reproducibility have not been rigorously examined. We applied MALDI-TOF MS to characterize a model system consisting of five methicillin-sensitive (MSSA) and five methicillin-resistant S. aureus isolates (MRSA) under two culture conditions (agar and broth) and using two sample preparation methods [intact cell method (ICM) and protein extraction method (PEM)]. The effects of these treatments on spectrum quality and reproducibility were quantified. PEM facilitated increases in the number of peaks and mass range width. Broth cultures further improved spectrum quality in terms of increasing the number of peaks. In addition, PEM increased reproducibility in samples prepared using identical culture conditions. MALDI imaging data suggested that the improvement in reproducibility may result from a more homogeneous distribution of sample associated with the broth/PEM treatment. Broth/PEM treatment also yielded the highest rate (96%) of correct classification for MRSA. Taken together, these results suggest that broth/PEM maximizes the performance of MALDI-TOF MS to characterize S. aureus. This article is protected by copyright. All rights reserved.

Concepts: Mass spectrometry, Staphylococcus aureus, Matrix-assisted laser desorption/ionization, Time-of-flight, Copyright, Michael Karas, Franz Hillenkamp

26

A new polyene macrolide antibiotic PN00053 was isolated from the fermentation broth of Streptomyces sp. wild-type strain MTCC-5680. The producer strain was isolated from fertile mountain soil of Naldehra region, Himachal Pradesh, India. The compound PN00053 was purified through various steps of chromatographic techniques and bio-activity guided fractionation followed by its characterization using physiochemical properties, spectral data ((1) H-NMR, (13) C-NMR, HMBC, HSQC, and COSY) and MS analysis. PN00053 exhibited broad spectrum in vitro antifungal activity against strains of Aspergillus fumigatus (HMR), A. fumigatus ATCC 16424, Candida albicans (I.V.), C. albicans ATCC 14503, C. krusei GO6, C. glabrata HO4, Cryptococcus neoformans, Trichophyton sp. as well as fluconazole resistant strains C. krusei GO3 and C. glabrata HO5. It did not inhibit growth of gram positive and gram-negative bacteria, displaying its specificity against fungi. This article is protected by copyright. All rights reserved.

Concepts: Bacteria, Microbiology, Fungus, Yeast, Cryptococcus neoformans, Candida albicans, Candidiasis, Chloramphenicol

26

The Listeria genus comprises 10 recognized species. L. monocytogenes causes listeriosis in humans and other animals primarily via contaminated food or animal feed. L. ivanovii causes listeriosis in animals and on rare occasions in humans. The identification of non-pathogenic species of Listeria in foods indicates that conditions exist that support the growth of pathogenic strains and is used to facilitate the implementation of control and prevention measures. This study shows the development and evaluation of a 5'exonuclease real-time PCR assay for the rapid identification of L. seeligeri, L. welshimeri, L. monocytogenes, L. ivanovii, L. grayi and L. innocua. The assay consists of two triplexes which were evaluated using fifty-three cultures of Gram-positive bacteria, including 49 Listeria spp. from human, animal, food or food processing environments. The assay was rapid, specific and reproducible and could identify each of the six species from a mixture of strains. The developed assay proved to be a powerful means of rapidly identifying Listeria species and could be usefully implemented in busy specialist reference laboratories. This article is protected by copyright. All rights reserved.

Concepts: Bacteria, Polymerase chain reaction, Microbiology, Identification, Listeria monocytogenes, Listeria, Listeriosis, Gram-positive bacteria