More Cercospora Species Infect Soybeans across the Americas than Meets the Eye

Abstract

Diseases of soybean caused by Cercospora spp. are endemic throughout the world's soybean production regions. Species diversity in the genus Cercospora has been underestimated due to overdependence on morphological characteristics, symptoms, and host associations. Currently, only two species (Cercospora kikuchii and C. sojina) are recognized to infect soybean; C. kikuchii causes Cercospora leaf blight (CLB) and purple seed stain (PSS), whereas C. sojina causes frogeye leaf spot. To assess cryptic speciation among pathogens causing CLB and PSS, phylogenetic and phylogeographic analyses were performed with isolates from the top three soybean producing countries (USA, Brazil, and Argentina; collectively accounting for ~80% of global production). Eight nuclear genes and one mitochondrial gene were partially sequenced and analyzed. Additionally, amino acid substitutions conferring fungicide resistance were surveyed, and the production of cercosporin (a polyketide toxin produced by many Cercospora spp.) was assessed. From these analyses, the long-held assumption of C. kikuchii as the single causal agent of CLB and PSS was rejected experimentally. Four cercosporin-producing lineages were uncovered with origins (about 1 Mya) predicted to predate agriculture. Some of the Cercospora spp. newly associated with CLB and PSS appear to represent undescribed species; others were not previously reported to infect soybeans. Lineage 1, which contained the ex-type strain of C. kikuchii, was monophyletic and occurred in Argentina and Brazil. In contrast, lineages 2 and 3 were polyphyletic and contained wide-host range species complexes. Lineage 4 was monophyletic, thrived in Argentina and the USA, and included the generalist Cercospora cf. flagellaris. Interlineage recombination was detected, along with a high frequency of mutations linked to fungicide resistance in lineages 2 and 3. These findings point to cryptic Cercospora species as underappreciated global considerations for soybean production and phytosanitary vigilance, and urge a reassessment of host-specificity as a diagnostic tool for Cercospora.

Fig 1. Bayesian phylogeny (consensus tree) showing the relationships among causal agents of Cercospora leaf blight and purple seed stain of soybean.

The dataset was 3615 bases long and resulted from the concatenation of seven nuclear genes (act, cal, his, ITS, tef, cfp, and tub). Cercospora sojina is shown for reference purpose; the tree was rooted in C. beticola. Branch lengths are drawn to scale; nodal support values are given as posterior probabilities (%) above the branches (when ≥85%). Scale bar corresponds to the expected number of substitutions per site. Color in terminals according to the origin of the isolates: blue, Argentina; green, Brazil; red, United States. Color in branches according to lineage: blue, lineage 1; green, lineage 2; orange, lineage 3; and red, lineage 4. Black arrow indicates the ex-type strain of C. kikuchii (CPC_5068). Gene codes: act, actin; cal, calmodulin; his, histone H3; ITS: internal transcribed spacers and intervening 5.8S nrDNA; tef, translation elongation factor 1-alpha; cfp, cercosporin facilitator protein; tub, β-tubulin 1.

Fig 2. Bayesian phylogeny (consensus tree) showing the relationships among causal agents of Cercospora leaf blight and purple seed stain of soybean and other closely related species.

Fungal isolates were obtained from either purple-stained soybean seeds or infected leaves. The dataset was1566 bases long and resulted from the concatenation of five nuclear genes (act, cal, his, ITS, and tef). Branch lengths are drawn to scale; nodal support values are given as posterior probabilities (%) above the branches (when > 85%). Scale bar corresponds to the expected number of substitutions per site. Color in terminals according to the origin of the isolates: blue, Argentina; green, Brazil; red, United States; and black, data from 6]. Color in branches according to lineage: blue, lineage 1; green, lineage 2; orange, lineage 3; and red, lineage 4. Black arrow indicates the ex-type strain of C. kikuchii (CPC_5068). Gene codes: act, actin; cal, calmodulin; his, histone H3; ITS, internal transcribed spacers and intervening 5.8S nrDNA; tef, translation elongation factor 1-alpha.

Fig 3. Maximum clade credibility tree for seven nuclear genes of the causal agents of Cercospora leaf blight and purple seed stain of soybean.

Scale of the x axis is ages in millions of years (Mya). Cercospora sojina is shown for reference purpose; the tree was rooted in C. beticola. Color in terminals according to the origin of the isolates: blue, Argentina; green, Brazil; and red, United States. Color in branches according to lineage: blue, lineage 1; green, lineage 2; orange, lineage 3; and red, lineage 4. Black arrow indicates the ex-type strain of C. kikuchii (CPC_5068). Nodal support values are given as posterior probabilities (%) above the branches. Blue bars at nodes are the 95% highest probability density (HPD) intervals for the age of that node.

Fig 4. Median-joining networks.

Haplotype relationships are depicted within each of the four genealogical lineages of the causal agents of Cercospora leaf blight and purple seed stain of soybean. Networks were based on a dataset containing two nuclear gene regions concatenated: the cercosporin facilitator protein (cfp) and the β-tubulin 1 (tub) genes. In each network, a circle represents a given haplotype (coded with numbers); size is proportional to the relative frequencies. Numbers of mutational steps are indicated with bars when there is more than one (unless indicated otherwise). Color codes according to the origin of the sequences: blue, Argentina; green, Brazil; and red, United States. Diamonds indicate haplotypes harboring the mutation E198A, which is known to confer resistance to benzimidazole fungicides. In Lineage 1, an arrow denotes the location of the haplotype of CPC 5068, the ex-type strain of C. kikuchii.

Fig 5. Median-joining network depincting genealogical relationships among mtDNA haplotypes of Cercospora that is associated with purple seed stain in soybeans.

The network was based on a dataset (653 bases long) obtained from the mitochondrial cytochrome b (cyb) gene. Each circle represents a given haplotype (coded with letters); a line linking two haplotypes indicates a single mutation step. Color slices represent the frequencies of that haplotype in each of the four lineages (see Fig 3). NR, isolates harboring unexpected cfp–tub gene combinations in the nuclear genome. Numbers in parenthesis refer to the number of occurrences of that haplotype in Argentina, Brazil, and USA, respectively. Diamonds indicate haplotypes harboring the mutation A143G, which is known to confer resistance to Qoi inhibitor fungicides. Dashed line indicates network homoplasy owing to multiple, independent origins of mutation A143G.