fungal cells from fresh samples were mounted on a glass slide with a drop of water , and their structures were examined using bright - field and differential interference - contrast light microscopy . for measurements , an olympus bx51 microscope ( olympus , tokyo , japan ) and for imaging , a zeiss ax10 microscope equipped with an axiocam mrc5 ( carl zeiss , gttingen , germany ) were used . the representative specimen was submitted to the korea university herbarium ( kus ) under accession number kus - f27688 . to obtain a pure isolate , spores were collected from infected leaf tissues by using a sterile needle under a dissecting microscope and were placed on a drop of sterile water on a glass slide . a conidial suspension was streaked onto 2% water agar plates supplemented with 100 mg / l of streptomycin sulfate , and the plates were incubated at 25 for 4 days . suitable germinated conidia were transferred to potato dextrose agar ( pda ) plates using a sterile needle under a dissecting microscope . two monoconidial isolates were deposited in the korean agricultural culture collection , rural development administration , wanju , korea ( accession nos . kacc47656 and kacc48077 ) . after 7 days of incubation at 25 on pda plates in darkness , erumpent fungal colonies were formed with moderate aerial mycelia and smooth , lobate margins . the surface appearance was pale olivaceous - grey ; reverse iron - grey ( fig . stromata were not present or were weakly developed , subimmersed , globular , grayish dark brown , 20100 m , and visible as circular patches ( fig . conidiophores were fasciculate , brown to pale brown in color , straight to curved , smooth , unbranched , apex rounded to truncate , 1535  34 m , and aseptate to septate ( fig . conidiogenous cells were integrated , terminal , unbranched , brown to pale brown in color , smooth , and proliferating sympodially . conidia were solitary , gray to subhyaline , guttulate , obconically truncate at the base , obtuse at the apex , 3- to 6-septate , non - constricted at the septa , 2560  2.53.5 m , and had un - thickened and not darkened hila ( fig . these morphological characteristics were close to those of pseudocercospora pyricola ( sawada ) j. m. yen   and pseudocercospora photiniae ( fukui ) c. nakash . & tak . kobay . , which have been recorded on several rosaceous trees and share common features with respect to conidiophores and conidia , suggesting that they might be synonymous . since the former species is an older name , we concluded that the korean isolate should be identified as p. pyricola ( sawada ) j. m. yen . genomic dna was extracted from harvested mycelia from the cultures grown on pda ( kacc47656 ) using a dneasy plant mini kit ( qiagen inc . , valencia , ca , usa ) . primers employed for the amplification of ef-1 included ef1 - 728f   and ef-2 , while act-512f   and act-2rd   were used to amplify a portion of the act gene . the complete its region of ribosomal dna was amplified using primers its-4 and its-5 , while primers used for amplification of the 28s lsu region were lsu1fd   and lr5 . the amplified pcr products were separated on a 1.5% agarose gel , followed by purification with a pcr purification kit ( core - one ; core - bio , seoul , korea ) . both amplicon strands were sequenced using the same primers that were used for the initial amplification . the reactions were monitored using bigdye terminator cycle sequencing kits ( applied biosystems , foster city , ca , usa ) as indicated by the manufacturer and were analyzed on an abi 3130 automated dna sequencer ( applied biosystems ) . the possible identity of the isolates was established by comparing their its , lsu , ef-1 , and act sequences with those in the genbank database ( national center for biotechnology information [ ncbi ] , us national institute of health , bethesda , md , usa ; http://www.ncbi.nlm.nih.gov/blast ) . selected pseudocercospora sequences , including ef-1 , act , its , and lsu , were retrieved from genbank for the phylogenetic analysis . these retrieved sequences were included in the pseudocercospora phylogenetic tree constructed by crous et al . . the obtained sequences were edited and assembled using the seqman software ( lasergene ; dnastar , madison , wi , usa ) . a neighbor - joining phylogenetic tree was constructed using the maximum composite likelihood method by mega6 . the representative and resulting 490-bp ef-1 , 578-bp act , 525-bp its , and 861-bp lsu sequences obtained from kacc47656 were deposited in genbank ( accession nos . ky048164 , ky048162 , ky048161 , and ky048163 ) . a blast search in genbank using the its sequence revealed that the sequence showed over 99% identity with several sequences of pseudocercospora species , including p. paraquayensis , p. neriicola , p. lilacis , and p. rhododendrigena . however , the ef1- sequences varied between these species , even though they had the same its , act , and lsu sequences . the phylogenetic tree , created using a dataset of four combined sequences , translation elongation factor 1 ( tef-1 ) , act , its , and lsu , showed that p. pyricola from black chokeberry was included in a well - supported clade consisting of pseudocercospora spp . ( bootstrap value of 100% ) that was distinct from other genera , such as passalora , pallidocercospora , pseudocercosporella , septoria , and cercospora ( fig . tests in accordance with koch 's postulates were carried out to establish the pathogenicity of the isolated fungus . a conidial suspension with mycelial fragments ( approximately 1 10 propagules / ml ) of isolate kacc47656 was prepared using a 7-day - old culture grown on v8 juice agar by flooding the plates with sterilized water . the mixed suspension was sprayed onto the young leaves of five healthy 2-year - old plants (  nero ) until it began to run off . five control plants were sprayed with sterilized water . treated and control plants were individually covered with polythene bags to maintain 100% relative humidity for 24 hr and then were transferred to separate compartments and maintained at 28  2 with high ( > 80% ) relative humidity in a greenhouse . typical leaf spots were observed on the inoculated plants after 14 days of incubation , and were identical to those observed in the field . the fungus that was re - isolated from the lesions of the inoculated plants was identical to the original isolate and confirmed as p. pyricola . leaf spots caused by p. pyricola have previously been reported on several species of pyrus ( rosaceae ) , such as p. communis ( china ) , p. lindleyi ( china ) , p. serotina ( taiwan ) , p. serrulata ( china ) , and pyrus sp . furthermore , these reports relied mainly on a combination of morphological characteristics and host specificity without any studies of molecular characteristics . to date , cercospora leaf spots which have been reported on black chokeberry worldwide have been known to be caused by cercospora pyri ( usa ) , cercosporella pirina ( canada , usa ) , and cercosporella pyri ( canada , usa ) . however , morphological and molecular characteristics of the fungus isolated from black chokeberry were not consistent with the species of cercospora and cercosporella . as shown above , this fungus is morphologically close to p. pyricola and p. photiniae . although we concluded that this fungus should be identified as p. pyricola over p. photiniae based on morphological characteristics and taxonomic priority , further studies on the complexity of pseudocercospora spp . on rosaceous host plants should be conducted . to our knowledge , this is the first report on p. pyricola infection of black chokeberry . since the sequences of p. pyricola and p. photiniae have never appeared in phylogenetic studies , this study provides a good platform for future studies on the taxonomy of pseudocercospora and further investigations into pseudocercospora - black chokeberry associations . our observations in several orchards showed that pseudocercospora leaf spots expanded rapidly on young leaves and detracted from the yield value of fruit .
leaf spot disease on black chokeberry ( aronia melanocarpa ) was observed at several locations in korea during 20142015 . 
 leaf spots were distinct , scattered over the leaf surface and along the leaf border , subcircular to irregular and brown surrounded by a distinct dark color , and were expanded and coalesced into irregularly shaped lesions . 
 severely infected leaves became dry and fell off eventually . 
 the causative agent was identified as pseudocercospora pyricola . 
 morphological observations and phylogenetic analyses of multiple genes , including internal transcribed spacer , translation elongation factor 1-alpha , actin , and the large subunit ribosomal dna were conducted . 
 the pathogenicity test was conducted twice yielding similar results , fulfilling koch 's postulates . to our knowledge , this is the first report on p. pyricola infection of a. melanocarpa globally .