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Cercospora kikuchii (Matsumato & Tomoyasu) M.W. Gardner Proceedings of the Indiana Academy of Science (). Cercospora kikuchii (T. Matsumoto & Tomoy.) M.W. Gardner, Proc. Indiana Acad. Sci.: 12 () [MB#]. Caused by a fungal pathogen, Cercospora kikuchii. Infection is favored by humid conditions and temperatures of 75 to 80 F or higher. Can be found throughout.

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Conceived and designed the experiments: Diseases of soybean caused by Cercospora spp. 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. 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. From these analyses, the long-held assumption of C. Four cercosporin-producing lineages were uncovered with origins about 1 Mya predicted to predate agriculture. Some of the Cercospora spp.

Lineage 1, which contained the ex-type strain of C. 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. 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.

Due to a variety of environmental and genetic constraints, the ability of a pathogen to cause disease in a host plant is not the rule but an exception. Nevertheless, historical records of major disease outbreaks, such as the potato blight that caused the infamous Irish famine of the mids, serve as reminders of the relentless power of plant pathogens.

One of the key elements for understanding how plant diseases originate, disseminate, and can be managed is the proper identification of their causal agents. The cosmopolitan genus Cercospora Fresen.

Mycosphaerellaceae, Ascomycota contains some of the most destructive plant pathogens. Within Cercosporamore than 3, species have been described [ 1 ], although species are currently recognized; morphologically indistinguishable species have been lumped and referred to as C.

Cercospora kikuchii

The lack of a consistent array of diagnostic characters makes species identification in Cercospora a daunting task. For most Cercospora spp. Traditionally, anamorphic members of Cercospora have been identified to species based on the morphology of conidia and conidiophores, and on the belief that most species of Cercospora are strictly host-specific [ 4 — 6 ], even to the extent that new species have been described solely based upon occurrence in a distinct host [ 6 ].

With the advent of molecular tools, a new theme has emerged in Cercospora taxonomy: According to the current knowledge, there are only two species of Cercospora that infect soybeans Glycine max [L. Gardner; both are believed to be host specific [ 9 ]. Despite the difference, a previous phylogenetic study placed both C. Identification of this pathogen relies heavily on the classical symptoms of PSS: The purple discoloration is caused by the accumulation of cercosporin, which causes membrane damage and cell death [ 13 ], subsequently decreasing grain marketability and seedling vigor [ 14 ].

Cercospora leaf blight causes significant yield loss when blighting leads to defoliation at the time pods are filling [ 15 ].

The distribution of C. Besides, there were conflicting reports on isolate-dependent conditions for sporulation in vitro [ 21 ], and phenotypic variation among axenic cultures has been observed [ 1822 ]. A large number of vegetative compatibility groups were uncovered in C. Considering that this species has arrived relatively recently in the Americas owing to the recent introduction of soybean cultivation, these variations seem unusually large.


From a practical perspective, management of soybean diseases has relied heavily on applications of broad-spectrum chemical fungicides benzimidazoles and strobilurins, either alone or in combination e. Applications of these fungicides may not be intended to control either CLB or PSS directly; nevertheless, the causal agents are often exposed to strong chemical selective pressures, and thus resistance may emerge over time.

Some point mutations are known to confer fungicide tolerance. Then, we used an array of complementary phylogenetic and phylogeographic tools to address the following four questions: Field studies did not involve endangered or protected species. No collecting permits were required as these are introduced, alien plant pathogens of cosmopolitan occurrence. When infested soybeans were collected in the field, the owner’s permission was obtained.

Fungal isolates used in this study are part of personal culture collections of L.

Oliveira BrazilE. Guillin Argentinaand B. Bluhm USA and are maintained at their respective institutions.

Cercospora Leaf Blight and Purple Seed Stain of Soybeans

The cultures were obtained from either infected soybean leaves or purple stained seeds; isolation techniques followed protocols that were described elsewhere [ 61822 ]. Fungal mycelia were collected from cultures grown on potato dextrose agar PDA [ 18 ]. Polymerase chain reactions PCR were performed to amplify seven nuclear gene regions and the mitochondrial cyb gene region. The seven nuclear gene regions included the five regions previously used to obtain a comprehensive phylogeny of Cercospora [ 6 ].

The protocols, primers, and PCR conditions to amplify actcalhisITS, and tef were essentially those described previously [ 6 ]. For the amplification of the cyb gene region, we followed the instructions of a previous study [ 28 ]. Detailed information on primer design and usage is provided S1 Table. Amplification of the cfp gene region required the design of new primers, which was accomplished by analyzing the cfp gene of C.

The following two primer pairs were designed: The amplifications used ng of genomic DNA, 0. Amplification of the tub gene region targeted two non-overlapping fragments. Sequencing services were performed by Macrogen Inc. Sequencing reactions were carried out with the same primers as in the PCR amplifications. All sequences were imported into Sequencher version 4. After all of the sequences were aligned, the ends of the alignments were trimmed to eliminate fragments that could not be obtained for all sequences.

Sequences obtained for this study were deposited in GenBank S2 Table. Preliminary analyses showed that the cfp and tub sequences each fell within one of four distinct subsets of sequences. Therefore, we prepared a concatenated cfp — tub alignment and searched for the presence of intergenic recombinants, that is, isolates that could harbor unexpected cfp — tub gene combinations.

We then scanned the datasets of both cfp and tub independently with the program RDP3 [ 31 ] to search for intragenic recombination events using seven different recombination detection methods. Multiple datasets were assembled throughout the study to accommodate the particular requirements of each analysis. Sequences for the remaining five nuclear genes act jikuchii, calhisITS, and tef came from the isolate CPCwhich is the strain type of C.

The six sequences of C. In addition to C. This dataset was intended to explore the phylogenetic relationships among species of Cercospora that infect soybeans. Because sequences of cfp and tub were not available for kikuchil species, we restricted this dataset to sequences of the five nuclear cefcospora act, calhisITS, and tef [ 6 ] together with our own set of corresponding sequences. The sequences belonged to 18 recognized species of Cercosporawith C.


Dataset S2 aimed to uncover phylogenetic relationships among soybean-infecting Cercospora and other congeneric species within a broader kikuchij of the genus that has been recently published [ 6 ].

For tubwe split the sequence information into exons TUBexon and introns TUBintron and considered each subset as a separate partition. This dataset was used for time divergence estimates. We also assembled five additional datasets that were intended for network analyses. Four datasets were created containing the concatenated sequences of the cfp and tub genes; there was one dataset for each subset harboring the expected cfp — tub combination: In datasets S4 to S8, each indel, regardless of its size, was considered a fifth state and coded as a single mutation.

The phylogenetic relationships among the species of Kikucjii were inferred by means of Bayesian phylogeny. For each gene region, the Akaike Information Kokuchii [ 33 ] indicated the best-fit models among 24 models of molecular evolution as following: The analysis was carried out using two simultaneous runs of five million kikcuhii each, with one cold and seven heated chains in each run; the temperature parameter was set to 0.

Trees were sampled once every 5, generations. A second phylogenetic analysis investigated the relationships of Cercospora and 18 closely-related, congeneric species.

This analysis was carried out using two simultaneous runs of 20 million generations each, with one cold and seven heated chains in each run; the temperature parameter was set to 0. Trees kjkuchii sampled once every 20, generations. In the preceding analyses, the kikuhii trees were kikuuchii as burn-in samples; average standard deviation of split frequencies at the end of each run was below 0.

The selected settings ensured sufficient sampling of the posterior occurred; in Tracer 1. The third phylogenetic analysis compared the information we have gathered in our collection of isolates with the results of a previous investigation on molecular diversity of Kiikuchii.

The analysis was carried out using two simultaneous runs of one million generations each, with one cold and seven heated chains in each cercosproa the temperature parameter was set to 0. Trees were sampled once every 1, generations; the first trees were discarded as burn-in samples; average standard deviation of split frequencies at the end of each run was 0.

In this input file, xercospora substitution models and clock models were unlinked. For each gene partition, we implemented HKY as the model of molecular evolution. As no fossil records exist to calibrate the phylogeny, we relied upon the molecular clock hypothesis and used published mutation rates for the exons and introns of the tubulin gene as previously calculated [ 36 ].

Kkkuchii these rates, introns mutate 4. The analysis was carried out under the strict molecular clock assumption. A Yule speciation process model was selected for tree prior.

The analysis was run for 10 million generations, with samples taken every These kjkuchii ensured that both model parameters and time estimates were sampled adequately, as the ESS values were above for all statistics in Tracer 1. To infer genetic connections in the network analyses, we used information from the concatenated sequences of the nuclear cfp and tub genes datasets S4 to S7 and the mitochondrial cyb gene dataset S8. Measures of molecular diversity number of haplotypes, H; haplotype diversity, H d ; nucleotide diversity, pi; average number of nucleotide diversity, k; and number of variable sites, S were estimated in DNAsp v5 [ 38 ].