Effect of streptomycin treatment on bacterial community structure in the apple phyllosphere

We studied the effect of many years of streptomycin use in apple orchards on the proportion of phyllosphere bacteria resistant to streptomycin and bacterial community structure. Leaf samples were collected during early July through early September from four orchards that had been sprayed with streptomycin during spring of most years for at least 10 years and four orchards that had not been sprayed. The percentage of cultured phyllosphere bacteria resistant to streptomycin at non-sprayed orchards (mean of 65%) was greater than at sprayed orchards (mean of 50%) (P = 0.0271). For each orchard, a 16S rRNA gene clone library was constructed from leaf samples. Proteobacteria dominated the bacterial communities at all orchards, accounting for 71 of 104 OTUs (determined at 97% sequence similarity) and 93% of all sequences. The genera Massilia, Methylobacterium, Pantoea, Pseudomonas, and Sphingomonas were shared across all sites. Shannon and Simpson's diversity indices and Pielou's evenness index were similar among orchards regardless of streptomycin use. Analysis of Similarity (ANOSIM) indicated that long-term streptomycin treatment did not account for the observed variability in community structure among orchards (R = -0.104, P = 0.655). Other variables, including time of summer, temperature and time at sampling, and relative distance of the orchards from each other, also had no significant effect on bacterial community structure. We conclude that factors other than streptomycin exposure drive both the proportion of streptomycin-resistant bacteria and phylogenetic makeup of bacterial communities in the apple phyllosphere in middle to late summer.