Molecular detection of Gluconacetobacter sacchari associated with the pink sugarcane mealybug Saccharicoccus sacchari (Cockerell) and the sugarcane leaf sheath microenvironment by FISH and PCR (original) (raw)
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International Journal of Systematic and Evolutionary Microbiology, 1999
Bureau of Sugar A new species of the genus Gluconacefobacfer, for which the name Gluconacefobacfer sacchari sp. nov. is proposed, was isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug, Saccharicoccus sacchari, found on sugar cane growing in Queensland and northern New South Wales, Australia. The nearest phylogenetic relatives in the a-subclass of the Pro f eobacferia a re Glucona ce f obacter lique faciens and Gluconace tobacf er diazotrophicus, which have 988-993 O/ O and 97.9-98-5 O/ O 165 rDNA sequence similarity, respectively, to members of Gluconacetobacfer sacchari. On the basis of the phylogenetic positioning of the strains, DNA reassociation studies, phenotypic tests and the presence of the Q l O ubiquinone, this new species was assigned to the genus Gluconacefobacfer. No single phenotypic characteristic is unique to the species, but the species can be differentiated phenotypically from closely related members of the acetic acid bacteria by growth in the presence of 0 0 1 YO malachite green, growth on 30% glucose, an inability to f i x nitrogen and an inability to grow with the L-amino acids asparagine, glycine, glutamine, threonine and tryptophan when D-mannitol was supplied as the sole carbon and energy source. The type strain of this species is strain SRI 1794T (= DSM 127173.
Members of the genus Saccharomonospora are isolated infrequently, probably due to the low occurrence of these actinomycetes in the environment. Although members of this genus can easily be identified by micromorphological criteria, the extensive chemotaxonomic characterization of each new isolate is a time-consuming task which cannot always be undertaken when handling large numbers of strains as is the case in natural products screening programmes. In this work, the design of one set of genusspecific oligonucleotides which allows rapid detection of members of the genus Saccharomonospora by means of PCR-specific amplification is presented. The genus specificity of these primers was validated on a wide range of collection and wild-type strains, and subsequently applied to evaluate the presence of representatives of this taxon directly from soil DNAs. Partial 16S rDNA sequencing of representative wild-type strains was used to validate their genus assignment. Further analyses of PCR fingerprinting patterns and 16S-23S internal transcribed spacer sequences were used to determine the diversity of wild-type isolates obtained from soils. This study shows the usefulness of the application of these primers for the direct identification of members of this genus and in assessment of its occurrence within natural microbial habitats.
Journal of Microbiological Methods, 1996
A multiplex PCR telzhnique for the rapid identification of Succharomonospora strains was developed. Primers for the four validly described Succharomonosporu species were designed by aligning previously published 16s rRNA sequences. These primers were found to be species-specific through the application of a specificity test. Four species-specific primers were added to a single react Ion tube together with a universal reverse primer corresponding to the 3' terminus of 16s rRNA genes.
African Journal of Microbiology Research, 2014
The objective of this study was to evaluate the viability of real-time polymerase chain reaction (PCR) to detect Gluconacetobacter diazotrophicus in sugarcane inoculated and non-inoculated with diazotrophs which are grown under field conditions. The primer pair PAL5F and PAL5R yielded a specific band of 189 bp using real time PCR with SYBER Green I. This primer pair was the most sensitive one to detect endophytic bacteria in sugarcane plants grown under field conditions and inoculated or not with bacterium. The lower limit of detection was 5 fg of template DNA, which corresponds to 12 bacterial cells. In contrast, a cultivation-dependent approach was not capable of detecting the bacteria in the same sample. The quantification of G. diazotrophicus from field grown plants using real-time PCR and a set of specific primers can be used to determine the number of bacterial cells that colonize endophytically the plant after inoculation. A highly sensitive and specific assay was developed to quantify G. diazotrophicus in sugarcane plants grown under field conditions. This assay can be used to evaluate the occurrence of the bacterium in different sample types.
Identification of Saccharomonospora Strains by the Use of Genomic DNA Fragments and rRNA Gene Probes
Restriction digestion fragments of DNAs extracted from 14 representative strains of Saccharomonospora azurea, "Saccharomonospora caesia ," Saccharomonospora cyanea, Saccharomonospora glauca, and Saccharomonospora viridis and six "Saccharomonospora"-like isolates were separated by electrophoresis, Southern blotted onto nylon membranes, and hybridized by using two rRNA gene probes cloned from Streptomyces griseus subsp. griseus KCTC 9080. The following four restriction endonucleases were used: BarnHI, SaZI, PvuII, and XhoI. The resultant five ribotype patterns were considered species specific. The genomic diversity revealed by ribotyping indicates that this method can be used to both characterize and identify saccharomonosporae. All of the test strains contained DNA with three rRNA gene clusters.
Saccharibacillus sacchari gen. nov., sp. nov., isolated from sugar cane
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 2008
A bacterial strain designated GR21 T was isolated from apoplastic fluid of Saccharum officinarum (sugar cane). Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate forms a separate branch within the family 'Paenibacillaceae', with Paenibacillus as the closest related genus. Within this genus, the closest related species is Paenibacillus xylanilyticus, with 93.4 % similarity to the sequence of the type strain. The isolate has Gram-variable, facultatively anaerobic, rod-shaped cells, motile by polar and subpolar flagella. Round, non-ornamented, central or subterminal spores are formed in unswollen sporangia. The strain is catalase-positive and oxidase-negative on nutrient agar medium. Cellulose and aesculin were hydrolysed, whereas xylan, starch and gelatin were not. Growth was supported by many carbohydrates as carbon sources. Strain GR21 T displayed a lipid profile consisting of diphosphatidylglycerol, phosphatidylglycerol, an unknown aminophospholipid, two unknown glycolipids and an unknown phosphoglycolipid. MK-7 was the predominant menaquinone and anteiso-C 15 : 0 was the major fatty acid. The DNA G+C content was 57.8 mol%. Phylogenetic and phenotypic analyses, including assimilation of carbon sources and exoenzyme production commonly used for classification within the family 'Paenibacillaceae', showed that strain GR21 T belongs to a new genus within this family, for which the name Saccharibacillus sacchari gen. nov., sp. nov. is proposed. The type strain of Saccharibacillus sacchari is GR21 T (5LMG 24085 T 5DSM 19268 T ).
Microbes and Environments, 2009
Twenty-six strains of acetic acid bacteria were isolated from fruits, flowers and related materials collected in Thailand. They were divided into three genera, Acetobacter, Gluconobacter and Asaia, by phenotypic characterization and 16S rRNA gene sequence analyses. On the basis of 16S-23S rRNA gene internal transcribed spacer (16S-23S rDNA ITS) restriction and 16S rRNA gene sequence analyses, fourteen isolates assigned to the genus Acetobacter were divided into five groups: 1) Group 1A or A. tropicalis (one isolate); 2) Group 2A or A. orientalis (four isolates); 3) Group 3A or A. pasteurianus (five isolates); 4) Group 4A or A. syzygii (one isolate); and 5) Group 5A or A. ghanensis (three isolates). The eleven isolates assigned to the genus Gluconobacter were divided into three groups: 6) Group 1B or G. frateurii (four isolates); 7) Group 2B or G. japonicus (six isolates); and 8) Group 3B or unidentified (one isolate). The remaining isolate was placed into: 9) Group 1C or unidentified, which was assigned to the genus Asaia and considered to constitute a new species on the basis of the 16S rRNA gene sequence analysis and DNA-DNA hybridization.
INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 2000
DNA corresponding to 16S rDNA and the 16S-23S rDNA intergenic spacer (ITS) from 22 reference strains of acetic acid bacteria, representing the diversity of the family Acetobacteraceae, and 24 indigenous acetic acid bacteria isolated from wine fermentations were analysed by PCR-RFLP. Frateuria aurantia LMG 1558 T and Escherichia coli ATCC 11775 T were included as outgroups. PCRamplified products of about 1450 bp were obtained from the 16S rDNA of all the strains and products of between 675 and 800 bp were obtained from the 16S-23S rDNA ITS. PCR products were digested with 4-base-cutting restriction enzymes in order to evaluate the degree of polymorphism existing among these strains. Of the enzymes tested, TaqI and RsaI were the most discriminatory and showed no intraspecific variations in the restriction patterns. Restriction analysis of the 16S rDNA with these enzymes is proposed as a rapid and reliable method to identify acetic acid bacteria at the level of genus and species (or related species group) and its applicability to identification of indigenous acetic acid bacteria was demonstrated. The same degree of distinction as that for the 16S rDNA analysis was obtained within reference strains of acetic acid bacteria by PCR-RFLP of the 16S-23S rDNA ITS. However, 16S-23S rDNA ITS restriction patterns of strains isolated from wine did not match those of any of the reference strains. Thus, PCR-RFLP of the 16S-23S rDNA ITS is not a useful method to identify isolates of acetic acid bacteria at the species level, although it may be an adequate method to detect intraspecific differentiation.