Culture-dependent and culture-independent assessment of bacteria in the apple phyllosphere

Aims: Bacterial communities in the apple phyllosphere were examined quantitatively and qualitatively by applying culture-dependent and culture-independent methods. Methods and Results: Populations estimated by viewing cells stained with 4',6-diamidino-2-phenylindole generally were at least 100-1000 times greater than populations estimated by culturing on tryptic soy agar (TSA). Of the 44 operational taxonomic units (OTUs; cut-off threshold of 97%) detected in total, five bacterial orders containing 23 OTUs were identified by culturing on TSA, whereas nine orders containing 33 OTUs were identified by 16S rRNA gene cloning of DNA extracted from apple leaf surfaces. Twelve of the 44 OTUs were shared between cultured isolates and 16S rRNA gene clones and included the orders Burkholderiales, Pseudomonadales, Rhizobiales and Sphingomonadales. Three OTUs within the genus Sphingomonas accounted for 40% of isolates and 68% of clones. The Actinomycetales were found only among isolates, whereas the Bacteroidales, Enterobacteriales, Myxococales and Sphingobacteriales were represented in the 16S rRNA gene clone libraries but were absent among isolates. Conclusions: Culture-independent methods revealed greater numbers and greater richness of bacteria on apple leaves than found by culturing. Significance and Impact of the Study: This is the first study to directly compare culture-dependent and independent approaches for assessing bacterial communities in the phyllosphere. The biases introduced by different methods will have a significant impact on studies related to phyllosphere ecology, biological control of plant diseases, reservoirs of antibiotic resistance genes and food safety.