Novel primers improve species delimitation in Cercospora

Abstract

The genus <i>Cercospora</i> includes many important plant pathogens that are commonly associated with leaf spot diseases on a wide range of cultivated and wild plant species. Due to the lack of useful morphological features and high levels of intraspecific variation, host plant association has long been a decisive criterion for species delimitation in <i>Cercospora</i>. Because several taxa have broader host ranges, reliance on host data in <i>Cercospora</i> taxonomy has proven problematic. Recent studies have revealed multi-gene DNA sequence data to be highly informative for species identification in Cercospora, especially when used in a concatenated alignment. In spite of this approach, however, several species complexes remained unresolved as no single gene proved informative enough to act as DNA barcoding locus for the genus. Therefore, the aims of the present study were firstly to improve species delimitation in the genus <i>Cercospora</i> by testing additional genes and primers on a broad set of species, and secondly to find the best DNA barcoding gene(s) for species delimitation. Novel primers were developed for <i>tub2</i> and <i>rpb2</i> to supplement previously published primers for these loci. To this end, 145 <i>Cercospora</i> isolates from the Iranian mycobiota together with 25 additional reference isolates preserved in the Westerdijk Fungal Biodiversity Institute were subjected to an eight-gene (ITS, <i>tef1</i>, <i>actA</i>, <i>cmdA</i>, <i>his3</i>, <i>tub2</i>, <i>rpb2</i> and <i>gapdh</i>) analysis. Results from this study provided new insights into DNA barcoding in <i>Cercospora</i>, and revealed <i>gapdh</i> to be a promising gene for species delimitation when supplemented with <i>cmdA</i>, <i>tef1</i> and <i>tub2</i>. The robust eight-gene phylogeny revealed several novel clades within the existing <i>Cercospora</i> species complexes, such as <i>C. apii</i>, <i>C. armoraciae</i>, <i>C. beticola</i>, <i>C.</i> cf. <i>flagellaris</i> and <i>Cercospora</i> sp. G. The <i>C. apii s. lat.</i> isolates are distributed over three clades, namely <i>C. apii s. str.</i>, <i>C. plantaginis</i> and <i>C. uwebrauniana</i> sp. nov. The <i>C. armoraciae s. lat.</i> isolates are distributed over two clades, <i>C. armoraciae s. str.</i> and <i>C. bizzozeriana</i>. The <i>C. beticola s. lat.</i> isolates are distributed over two clades, namely <i>C. beticola s. str.</i> and <i>C. gamsiana</i>, which is newly described.