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

Molecular tools for the detection of the newly described acetic acid bacter ium Gluconacetobacter sacchari from the pink sugarcane mealybug, Saccharico ccus sacchari Cockerell (Homiptera: Pseudococcidae), and in the sugarcane l eaf sheath microenvironment were developed. G. sacchari specific 16S rRNA-t argeted 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 inter node scrapings. A sensitivity level of detection of 40-400 cells/reaction w as obtained using PCR from exponentially grown bacterial cultures and of 1- 10 cells in cane internode scrapings and leaf sheath fluid samples using ne sted PCR. The specificity of the primer set was demonstrated by the lack of amplification product formation in PCR by closely related acetic acid bact eria, including Gluconacetobacter liquefaciens, and Gluconacetobacter diazo trophicus. A Cy3 labeled probe for G. sacchari was designed and shown to be specific for the species. Investigation of the mealybug microenvironment b y whole cell fluorescent in situ hybridization revealed that G. sacchari ap pears to represent only a minor proportion of the population of the microbi ota in the mealybugs tested. This study has shown the usefulness of 16S rRN A-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. (C) 2000 Federation of European Microbiological Socie ties. Published by Elsevier Science B.V. All rights reserved.