scholarly journals Screening of Actinomycetes from Turmeric (Curcuma longa L.) and Ginger (Zingiber officinale) Rhizosphere for Antifungal Activity

2020 ◽  
pp. 18-28
Author(s):  
R. C. Osaro-Matthew ◽  
F. S. Ire ◽  
N. Frank-Peterside
Keyword(s):  
Antifungal Activity ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Culture Media ◽  
Curcuma Longa ◽  
Zingiber Officinale ◽  
Culture Technique ◽  
Rrna Gene ◽  
Dual Culture ◽  
Test Organisms

Aim: The present study was carried out to isolate actinomycetes from rhizospheric soil of Curcuma longa and Zingiber officinale and evaluate their antifungal potential. Methods: Actinomycetes were isolated from the rhizosphere soil of two Zingiberaceae plants (Curcuma longa and Zingiber officinale), using four different culture media. Isolates were screened for antifungal activity using dual culture technique against two reference strains Colletotrichum coccodes (DSM 2492) and Alternaria pimpriana (DSM 62023). The most potent strain was identified based on 16S rRNA gene sequence and the bioactive components of the strain were identified using GC-MS chromatography. Results: Fifteen strains of actinomycetes were isolated, SCA (starch casein agar) was found best for cultivation of actinomycetes. The 15 strains were grouped into three genera Norcadia 8(54%), Streptomyces 5(33%) and Janibacter 2(13%) based on morphological, biochemical and physiological identification. Among the 15 isolates 6(40%) strains showed activity against either of the test organisms, while 1(6.7%) was active against the both test organisms. Comparative analysis of the 16S rRNA gene sequences identified the potent isolates as Janibacter sp. strain RC18. GC-MS analysis revealed the presence of 20 compounds with 10 identified as potent antimicrobial metabolites. Conclusion: This study has revealed that rhizosphere of ginger and turmeric harbours rare actinomycetes that are valuable tool for sustainable agriculture.

scholarly journals Improved Amplification of Microbial DNA from Blood Cultures by Removal of the PCR Inhibitor Sodium Polyanetholesulfonate

1998 ◽  
Vol 36 (10) ◽  
pp. 2810-2816 ◽  
Author(s):  
David N. Fredricks ◽  
David A. Relman
Keyword(s):  
Blood Culture ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Benzyl Alcohol ◽  
Culture Media ◽  
Technical Problem ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Pcr Inhibitor ◽  
Purification Methods

Molecular methods are increasingly used to identify microbes in clinical samples. A common technical problem with PCR is failed amplification due to the presence of PCR inhibitors. Initial attempts at amplification of the bacterial 16S rRNA gene from inoculated blood culture media failed for this reason. The inhibitor persisted, despite numerous attempts to purify the DNA, and was identified as sodium polyanetholesulfonate (SPS), a common additive to blood culture media. Like DNA, SPS is a high-molecular-weight polyanion that is soluble in water but insoluble in alcohol. Accordingly, SPS tends to copurify with DNA. An extraction method was designed for purification of DNA from blood culture media and removal of SPS. Blood culture media containing human blood and spiked with Escherichia coli was subjected to an organic extraction procedure with benzyl alcohol, and removal of SPS was documented spectrophotometrically. Successful amplification of the extracted E. coli 16S rRNA gene was achieved by adding 5 μl of undiluted processed sample DNA to a 50-μl PCR mixture. When using other purification methods, the inhibitory effect of SPS could be overcome only by dilution of these samples. By our extraction technique, even uninoculated blood culture media were found to contain bacterial DNA when they were subjected to broad-range 16S rRNA gene consensus PCR. We conclude that the blood culture additive SPS is a potent inhibitor of PCR, is resistant to removal by traditional DNA purification methods, but can be removed by a benzyl alcohol extraction protocol that results in improved PCR performance.

scholarly journals Monitoring Bacterial Communities in Raw Milk and Cheese by Culture-Dependent and -Independent 16S rRNA Gene-Based Analyses

2007 ◽  
Vol 73 (6) ◽  
pp. 1882-1891 ◽  
Author(s):  
Céline Delbès ◽  
Leila Ali-Mandjee ◽  
Marie-Christine Montel
Keyword(s):  
16S Rrna ◽  
Culture Media ◽  
Raw Milk ◽  
Single Strand Conformation Polymorphism ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Plate Count ◽  
Rrna Gene ◽  
Dual Culture ◽  
Culture Dependent

ABSTRACT The diversity and dynamics of bacterial populations in Saint-Nectaire, a raw-milk, semihard cheese, were investigated using a dual culture-dependent and direct molecular approach combining single-strand conformation polymorphism (SSCP) fingerprinting and sequencing of 16S rRNA genes. The dominant clones, among 125 16S rRNA genes isolated from milk, belonged to members of the Firmicutes (58% of the total clones) affiliated mainly with the orders Clostridiales and the Lactobacillales, followed by the phyla Proteobacteria (21.6%), Actinobacteria (16.8%), and Bacteroidetes (4%). Sequencing the 16S rRNA genes of 126 milk isolates collected from four culture media revealed the presence of 36 different species showing a wider diversity in the Gammaproteobacteria phylum and Staphylococcus genus than that found among clones. In cheese, a total of 21 species were obtained from 170 isolates, with dominant species belonging to the Lactobacillales and subdominant species affiliated with the Actinobacteria, Bacteroidetes (Chryseobacterium sp.), or Gammaproteobacteria (Stenotrophomonas sp.). Fingerprinting DNA isolated from milk by SSCP analysis yielded complex patterns, whereas analyzing DNA isolated from cheese resulted in patterns composed of a single peak which corresponded to that of lactic acid bacteria. SSCP fingerprinting of mixtures of all colonies harvested from plate count agar supplemented with crystal violet and vancomycin showed good potential for monitoring the subdominant Proteobacteria and Bacteroidetes (Flavobacteria) organisms in milk and cheese. Likewise, analyzing culturable subcommunities from cheese-ripening bacterial medium permitted assessment of the diversity of halotolerant Actinobacteria and Staphylococcus organisms. Direct and culture-dependent approaches produced complementary information, thus generating a more accurate view of milk and cheese microbial ecology.

scholarly journals Antimicrobial Activity and Identification of the Biosynthetic Gene Cluster of X-14952B From Streptomyces sp. 135

2021 ◽  
Vol 12 ◽  
Author(s):  
Na Li ◽  
Simin Chen ◽  
Zhiqiang Yan ◽  
Jinhua Han ◽  
Yongquan Ta ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Antifungal Activity ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Cluster ◽  
Genome Mining ◽  
Biosynthetic Gene Cluster ◽  
Rrna Gene ◽  
Biosynthetic Gene ◽  
Type Strains

The bacterial genus Streptomyces is an important source of antibiotics, and genome mining is a valuable tool to explore the potential of microbial biosynthesis in members of this genus. This study reports an actinomycete strain 135, which was isolated from Qinghai-Tibet Plateau in China and displayed broad antimicrobial activity. The fermentation broth of strain 135 displayed strong antifungal activity (>70%) against Sclerotinia sclerotiorum, Botrytis cinerea, Valsa mali, Phytophthora capsici, Glomerella cingulata, Magnaporthe grisea, Bipolaris maydis, Exserohilum turcicum in vitro, meanwhile possessed significant preventive and curative efficacy against S. sclerotiorum, Gaeumannomyces graminis, and P. capsici on rape leaves (54.04 and 74.18%), wheat (90.66 and 67.99%), and pepper plants (79.33 and 66.67%). X-14952B showed the greatest antifungal activity against S. sclerotiorum and Fusarium graminearum which the 50% inhibition concentration (EC50) were up to 0.049 and 0.04 μg/mL, respectively. Characterization of strain 135 using a polyphasic approach revealed that the strain displayed typical features of the genus Streptomyces. 16S rRNA gene sequencing and phylogenetic analysis demonstrated that the isolate was most closely related to and formed a clade with Streptomyces huasconensis HST28T (98.96% 16S rRNA gene sequence similarity). Average nucleotide identity (ANI) and DNA-DNA hybridization (DDH) values in strain 135 and related type strains were both below the threshold of species determination (91.39 and 56.5%, respectively). OrthoANI values between strain 135 and related type strains are under the cutoff of determining species (<95%). The biosynthetic gene cluster (BGC) designated to X-14952B biosynthesis was identified through genome mining and the possible biosynthesis process was deduced.

scholarly journals Yania halotolerans gen. nov., sp. nov., a novel member of the suborder Micrococcineae from saline soil in China

2004 ◽  
Vol 54 (2) ◽  
pp. 525-531 ◽  
Author(s):  
Wen-Jun Li ◽  
Hua-Hong Chen ◽  
Ping Xu ◽  
Yu-Qin Zhang ◽  
Peter Schumann ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Culture Media ◽  
Phylogenetic Position ◽  
Rrna Gene ◽  
Optimum Growth Temperature ◽  
Gene Sequence Analysis ◽  
The Family ◽  
Nucleotide Data ◽  
Unknown Phospholipid

A novel coccoid, halotolerant actinobacterium, designated strain YIM 70085T, was isolated from a soil sample that was collected in Xinjiang Province, China, and characterized by using a polyphasic approach. Optimum growth temperature was 28 °C and growth occurred optimally in culture media that contained 10 % KCl. The peptidoglycan type was A4α, l-lys–gly–l-Glu. Whole-cell sugars consisted of xylose, mannose and galactose. Phospholipids were diphosphatidylglycerol, phosphatidylglycerol, one unknown phospholipid, one unknown glycolipid and traces of phosphatidylinositol. Menaquinones were MK-8 (83 %), MK-7 (12 %) and MK-9 (15 %). Predominant fatty acids were i-C15 : 0 (44·29 %), ai-C15 : 0 (35·60 %) and ai-C17 : 0 (9·74 %). The DNA G+C content was 53·5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 70085T occupies a branch that is distinct from, although very close to, the family Micrococcaceae in the suborder Micrococcineae. Based on its phenotypic characteristics, phylogenetic position (as determined by 16S rRNA gene sequence analysis) and 16S rDNA signature nucleotide data, it is concluded that the isolate represents a novel member of the suborder Micrococcineae, for which the name Yania halotolerans gen. nov., sp. nov. is proposed. The type strain is YIM 70085T (=CCTCC AA001023T=DSM 15476T).

scholarly journals Potential Role of Rhizobacteria Isolated from Citrus Rhizosphere for Biological Control of Citrus Dry Root Rot

Plants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 872
Author(s):  
Said Ezrari ◽  
Oumayma Mhidra ◽  
Nabil Radouane ◽  
Abdessalem Tahiri ◽  
Giancarlo Polizzi ◽  
...  
Keyword(s):  
Biological Control ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Root Rot ◽  
Rrna Gene ◽  
Dual Culture ◽  
Bacterial Isolates ◽  
Antimicrobial Substances ◽  
Plant Growth Promoting

Citrus trees face threats from several diseases that affect its production, in particular dry root rot (DRR). DRR is a multifactorial disease mainly attributed to Neocosmospora (Fusarium) solani and other several species of Neocosmospora and Fusarium spp. Nowadays, biological control holds a promising control strategy that showed its great potential as a reliable eco-friendly method for managing DRR disease. In the present study, antagonist rhizobacteria isolates were screened based on in vitro dual culture bioassay with N. solani. Out of 210 bacterial isolates collected from citrus rhizosphere, twenty isolates were selected and identified to the species level based on the 16S rRNA gene. Molecular identification based on 16S rRNA gene revealed nine species belonging to Bacillus, Stenotrophomonas, and Sphingobacterium genus. In addition, their possible mechanisms involved in biocontrol and plant growth promoting traits were also investigated. Results showed that pectinase, cellulose, and chitinase were produced by eighteen, sixteen, and eight bacterial isolates, respectively. All twenty isolates were able to produce amylase and protease, only four isolates produced hydrogen cyanide, fourteen isolates have solubilized tricalcium phosphate, and ten had the ability to produce indole-3-acetic acid (IAA). Surprisingly, antagonist bacteria differed substantially in their ability to produce antimicrobial substances such as bacillomycin (five isolates), iturin (ten isolates), fengycin (six isolates), surfactin (fourteen isolates), and bacteriocin (subtilosin A (six isolates)). Regarding the PGPR capabilities, an increase in the growth of the bacterial treated canola plants, used as a model plant, was observed. Interestingly, both bacterial isolates Bacillus subtilis K4-4 and GH3-8 appear to be more promising as biocontrol agents, since they completely suppressed the disease in greenhouse trials. Moreover, these antagonist bacteria could be used as bio-fertilizer for sustainable agriculture.

Detection of a novel bacterium associated with spores of the arbuscular mycorrhizal fungusGigaspora margarita

2009 ◽  
Vol 55 (6) ◽  
pp. 771-775 ◽  
Author(s):  
Liangkun Long ◽  
Qing Yao ◽  
Yuncan Ai ◽  
Mingrong Deng ◽  
Honghui Zhu
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Gene Sequence ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Mycorrhizal Fungus ◽  
Arbuscular Mycorrhizal ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Dual Culture ◽  
Rrna Gene Sequence

With PCR-denaturing gradient gel electrophoresis analysis, two bacterial 16S rRNA gene V3 region sequences, 7A and 7B, were detected in association with the crushed spores of the arbuscular mycorrhizal fungus Gigaspora margarita W.N. Becker & I.R. Hall 1976 MAFF520054. DNA sequencing and phylogenetic analysis revealed that 7B was mostly related to the documented cytoplasm endosymbiotic bacterium Candidatus Glomeribacter gigasporarum of G. margarita, but 7A could not be confidently assigned to a known taxon. Further characterization of 7A was conducted by obtaining its almost complete 16S rRNA gene sequence via PCR amplification and sequencing. BLAST search indicates that the 16S rRNA gene sequence did not match any identified species sequences in the GenBank database. Further detection revealed that 7A was also associated with the clean G. margarita MAFF520054 spores that were obtained by the surface-sterilized method or dual culture with Ri T-DNA transformed carrot roots. Many ellipse-shaped or egg-shaped bacterium-like organisms were clustered in layer 3 of the fungal spore wall by transmission electron microscopy observation. Our results indicate that 7A represents a novel bacterial population associated with G. margarita MAFF520054 spores, and its doubtless location (wall or cytoplasm) remains unclear based on the present data.

Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

2015 ◽  
Vol 41 (1) ◽  
pp. 51-58
Author(s):  
Mohammad Shamimul Alam ◽  
Hawa Jahan ◽  
Rowshan Ara Begum ◽  
Reza M Shahjahan
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fish Larva ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Valuable Insight ◽  
Multiple Sequence ◽  
Pcr Rflp ◽  
Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

Marine mammals are natural hosts of Oceanivirga salmonicida, a bacterial pathogen of Atlantic salmon

2020 ◽  
Vol 139 ◽  
pp. 161-174
Author(s):  
R Palmer ◽  
GTA Fleming ◽  
S Glaeser ◽  
T Semmler ◽  
A Flamm ◽  
...  
Keyword(s):  
16S Rrna ◽  
Atlantic Salmon ◽  
16S Rrna Gene ◽  
Marine Mammals ◽  
Rrna Gene ◽  
Bacterial Disease ◽  
Common Dolphin ◽  
Stenella Coeruleoalba ◽  
Striped Dolphin ◽  
Natural Hosts

During 1992 and 1993, a bacterial disease occurred in a seawater Atlantic salmon Salmo salar farm, causing serious mortalities. The causative agent was subsequently named as Oceanivirga salmonicida, a member of the Leptotrichiaceae. Searches of 16S rRNA gene sequence databases have shown sequence similarities between O. salmonicida and uncultured bacterial clones from the digestive tracts of marine mammals. In the current study, oral samples were taken from stranded dolphins (common dolphin Delphinus delphis, striped dolphin Stenella coeruleoalba) and healthy harbour seals Phoca vitulina. A bacterium with growth characteristics consistent with O. salmonicida was isolated from a common dolphin. The isolate was confirmed as O. salmonicida, by comparisons to the type strain, using 16S rRNA gene, gyrB, groEL, and recA sequence analyses, average nucleotide identity analysis, and MALDI-TOF mass spectrometry. Metagenomic analysis indicated that the genus Oceanivirga represented a significant component of the oral bacterial microbiomes of the dolphins and seals. However, sequences consistent with O. salmonicida were only found in the dolphin samples. Analyses of marine mammal microbiome studies in the NCBI databases showed sequences consistent with O. salmonicida from the common dolphin, striped dolphin, bottlenose dolphin Tursiops truncatus, humpback whale Megaptera novaeangliae, and harbour seal. Sequences from marine environmental studies in the NCBI databases showed no sequences consistent with O. salmonicida. The findings suggest that several species of marine mammals are natural hosts of O. salmonicida.

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