Molecular detection of Gluconacetobacter sacchari associated with the pink sugarcane mealybug Saccharicoccus sacchari (Cockerell) and the sugarcane leaf sheath microenvironment by FISH and PCR.

Abstract:

:Molecular tools for the detection of the newly described acetic acid bacterium Gluconacetobacter sacchari from the pink sugarcane mealybug, Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae), and in the sugarcane leaf sheath microenvironment were developed. G. sacchari specific 16S rRNA-targeted oligonucleotide primers were designed and used in PCR amplification of G. sacchari DNA directly from mealybugs, and in a nested PCR to detect low numbers of the bacteria from sugarcane leaf sheath fluid and cane internode scrapings. A sensitivity level of detection of 40-400 cells/reaction was obtained using PCR from exponentially grown bacterial cultures and of 1-10 cells in cane internode scrapings and leaf sheath fluid samples using nested PCR. The specificity of the primer set was demonstrated by the lack of amplification product formation in PCR by closely related acetic acid bacteria, including Gluconacetobacter liquefaciens, and Gluconacetobacter diazotrophicus. A Cy3 labeled probe for G. sacchari was designed and shown to be specific for the species. Investigation of the mealybug microenvironment by whole cell fluorescent in situ hybridization revealed that G. sacchari appears to represent only a minor proportion of the population of the microbiota in the mealybugs tested. This study has shown the usefulness of 16S rRNA-based molecular tools in the identification and detection of G. sacchari from environmental samples and will allow these tools to be used in further ecological research.

journal_name

FEMS Microbiol Ecol

authors

Franke IH,Fegan M,Hayward C,Leonard G,Sly LI

doi

10.1111/j.1574-6941.2000.tb00672.x

subject

Has Abstract

pub_date

2000-01-01 00:00:00

pages

61-71

issue

1

eissn

0168-6496

issn

1574-6941

pii

S0168-6496(99)00082-3

journal_volume

31

pub_type

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