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.

A red-shifted, mutated form of the jelly-fish green fluorescent protein (GFP) under control of a TEF promoter was expressed at high levels in the filamentous fungus Aureobasidium pullulans. In the three transformants studied, all morphotypes of the fungus, including pigmented chlamydospores, expressed GFP and fluoresced brightly. Confocal microscopy showed that the intra-cellular distribution of GFP was nonuniform. When applied to leaf surfaces, the transformants were readily visible and amenable to quantification by image analysis. Thus, GFP expression, together with quantitative image analysis, may provide a powerful method for ecological studies of plant-microbe relationships in nature.

Colonization of apple leaves by the yeast-like fungus Aureobasidium pullulans was studied in the field on eight dates over 2 years. Population densities from adaxial leaf surfaces were approximately log10 0.5-2.6 U higher when enumerated directly along line transects as microscopic counts of A. pullulans cells specifically identified by fluorescence in situ hybridization (FISH) than indirectly as CFU obtained by plating leaf washings from comparable surfaces onto nutrient media. Site-specific mapping of the leaf landscape colonized by A. pullulans was facilitated by use of geographic information system (GIS) software. Colonization was plotted and analyzed both qualitatively (as occupancy) and quantitatively (as density). Overall, when expressed as mean occupancy per date, 22-42% of the microscope fields (each 0.196 mm2) contained > or = 1 A. pullulans cell. Occupancy on a microscope field basis was greater over the midvein (47-89%) or smaller veins (49-65%) than over interveinal regions (11-21%). Intensity of colonization, whether expressed as percentage of total A. pullulans cells associated with a particular leaf feature or as cell density per unit area, was also significantly greater (P < 0.05) over the veinal areas compared with the interveinal areas. The primary fungal morphotypes involved in colonization were blastospores, swollen cells, and chlamydospores; only infrequently were hyphae or pseudohyphae seen. Numbers of microcolonies (> or = 10 clustered cells) and percentage of total A. pullulans cells that occurred as microcolonies increased over the growing season and were significantly greater (P < 0.05) over veinal regions compared with interveinal regions. Locally high concentrations of A. pullulans were associated with naturally occurring micro-wounds in interveinal areas. We conclude that A. pullulans colonizes the phylloplane predominantly as single cells and groups thereof in a highly heterogeneous fashion and that sites exist that are relatively conducive (veins; wounds) or nonconducive (unwounded interveinal areas) for epiphytic fungal growth.

Two strains of the dimorphic fungus Aureobasidium pullulans were grown in liquid and on solid media varying in carbon and nitrogen content, and on leaf surfaces. Hyphae were observed in all systems but comprised a very low proportion (often below quantitative detection) of the total biomass. In liquid media, hyphae were found sparsely and only in the wash-zone on walls of the culture flasks. Yeast phase growth (blastospores) occurred in pH-buffered media that were nutrient balanced, or continuously carbon-limited (fed-batch culture), or carbon-exhausted (batch culture). Blastospores exposed to conditions with limited nitrogen but sufficient organic carbon, or to acidified media, converted to swollen cells and chlamydospores. The latter morphotypes accumulated carbon internally as lipid granules, and then externally as capsular and soluble extracellular polysaccharide. They were cohesive and also adhered more strongly to cellulose membranes overlying agar media or to leaves than did blastospores. Pullulanase treatment diminished the capsules, cohesion, and adhesion. Addition of soluble extracellular polysaccharide to blastospores enhanced their adhesion to leaves. We conclude that extracellular polysaccharide can play a role in adhesion of A. pullulans.Key words: phylloplane, leaf surface, epiphytic growth, extracellular polysaccharide, cell attachment.

Gremmeniella abietina infects shoots of red and Scots pines through stomata on bracts th.it subtend short shoots. The gertn tulic penetrates between guard cells and sparsely colonizes bmct tissue by late summer or fall. Only after about late Januaiy or early Fcbruaiy of the following year does the fungus extend from the bract and begin eolonization of the short shoot and surrounding cortical tissue.~j