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Sooty blotch and flyspeck (SBFS) fungi on apple fruit were sampled from nine orchards in four midwestern U.S. states during 2000 and 30 orchards in 10 eastern U.S. states during 2005 in order to estimate taxonomic diversity and discern patterns of geographic distribution. Forty apple fruit per orchard were arbitrarily sampled and colonies of each mycelial phenotype were counted on each apple. Representative colonies were isolated, cultures were purified, and DNA was extracted. For representative isolates, the internal transcribed spacer (ITS) and large subunit (LSU) regions of ribosomal DNA were amplified and sequenced. In total, 60 SBFS putative species were identified based on ITS sequences and morphological characteristics; 30 of these were discovered in the 2005 survey. Modified Koch's postulates were fulfilled for all 60 species in an Iowa orchard; colonies resulting from inoculation of apple fruit were matched to the original isolates on the basis of mycelial type and ITS sequence. Parsimony analysis for LSU sequences from both surveys revealed that 58 putative SBFS species were members of the Dothideomycetes, 52 were members of the Capnodiales, and 36 were members of the Mycosphaerellaceae. The number of SBFS species per orchard varied from 2 to 15. Number of SBFS species and values of the Margalef and Shannon indexes were significantly (P < 0.05) lower in 21 orchards that had received conventional fungicide sprays during the fruit maturation period than in 14 unsprayed orchards. Several SBFS species, including Schizothyrium pomi, Pehaster fructicola, and Pseudocercosporella sp. RH 1, were nearly ubiquitous, whereas other species, such as Stomiopeltis sp. RS5.2, Phialophora sessilis, and Geastrumia polystigmatis, were found only within restricted geographic regions. The results document that the SBFS complex is far more taxonomically diverse than previously recognized and provide strong evidence that SBFS species differ in geographic distribution. To achieve more efficient management of SBFS, it may be necessary to understand the environmental biology of key SBFS species in each geographic region. [ABSTRACT FROM AUTHOR]