Young-Joon Choi

Evolution, Diversity, and Taxonomy of the Peronosporaceae, with Focus on the Genus Peronospora

Downy mildews are a notorious group of oomycete plant pathogens, causing high economic losses in various crops and ornamentals. The most species-rich genus of oomycetes is the genus Peronospora. This review provides a wide overview of these pathogens, ranging from macro- and micro-evolutionary patterns, their biodiversity and ecology to short overviews for the currently economically most important pathogens and potential emerging diseases. In this overview, the taxonomy of economically relevant species is also discussed, as the application of the correct names and species concepts is a prerequisite for effective quarantine regulations and phytosanitary measures.

Coupling Spore Traps and Quantitative PCR Assays for Detection of the Downy Mildew Pathogens of Spinach (Peronospora effusa) and Beet (P. schachtii)

Downy mildew of spinach (Spinacia oleracea), caused by Peronospora effusa, is a production constraint on production worldwide, including in California, where the majority of U.S. spinach is grown. The aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay for detection of airborne inoculum of P. effusa in California. Among oomycete ribosomal DNA (rDNA) sequences examined for assay development, the highest nucleotide sequence identity was observed between rDNA sequences of P. effusa and P. schachtii, the cause of downy mildew on sugar beet and Swiss chard in the leaf beet group (Beta vulgaris subsp. vulgaris). Single-nucleotide polymorphisms were detected between P. effusa and P. schachtii in the 18S rDNA regions for design of P. effusa- and P. schachtii-specific TaqMan probes and reverse primers. An allele-specific probe and primer amplification method was applied to determine the frequency of both P. effusa and P. schachtii rDNA target sequences in pooled DNA samples, enabling quantification of rDNA of P. effusa from impaction spore trap samples collected from spinach production fields. The rDNA copy numbers of P. effusa were, on average, ≈3,300-fold higher from trap samples collected near an infected field compared with those levels recorded at a site without a nearby spinach field. In combination with disease-conducive weather forecasting, application of the assays may be helpful to time fungicide applications for disease management.

Host Jumps and Radiation, Not Co‐Divergence Drives Diversification of Obligate Pathogens. A Case Study in Downy Mildews and Asteraceae

Even though the microevolution of plant hosts and pathogens has been intensely studied, knowledge regarding macro-evolutionary patterns is limited. Having the highest species diversity and host-specificity among Oomycetes, downy mildews are a useful a model for investigating long-term host-pathogen coevolution. We show that phylogenies of Bremia and Asteraceae are significantly congruent. The accepted hypothesis is that pathogens have diverged contemporarily with their hosts. But maximum clade age estimation and sequence divergence comparison reveal that congruence is not due to long-term coevolution but rather due to host-shift driven speciation (pseudo-cospeciation). This pattern results from parasite radiation in related hosts, long after radiation and speciation of the hosts. As large host shifts free pathogens from hosts with effector triggered immunity subsequent radiation and diversification in related hosts with similar innate immunity may follow, resulting in a pattern mimicking true co-divergence, which is probably limited to the terminal nodes in many pathogen groups.

Plasmopara invertifolia sp. nov. causing downy mildew on Helichrysum bracteatum (Asteraceae)

Plasmopara invertifolia sp. nov. causes severe leaf distortion and necrosis on Helichrysum bracteatum, a beautiful and important ornamental plant for trade in Brazil. This oomycete pathogen is distinguished from other species of Plasmopara on Asteraceae by its smaller sporangia and larger sporangiophores, which justifies the proposition of a new taxon in the genus Plasmopara to accommodate it. The phylogenetic analysis of cox2 gene sequence data supports such placement and also shows that P. invertifolia is close to the P. halstedii complex. Plasmopara invertifolia is then described, illustrated and discussed.

Characterisation and risk assessment of the emerging Peronospora disease on Aquilegia

Aquilegia is a popular garden plant in the northern hemisphere as well as a native plant in the UK and continental Europe. In 2000, Semiaquilegia adoxoides was found infected by downy mildew in Korea, and since 2013 there have been several confirmed records of a Peronospora sp. affecting Aquilegia in the UK, with symptomatic plants being observed several years prior. Symptoms include yellow patches delineated by the veins on the leaves of affected plants, but systemically infected plants were also recorded, which are generally chlorotic and show curled leaf margins. A greyish down of conidiophores and conidia was observed on the lower side of infected leaves. Preliminary molecular phylogenetic analyses could not identify the causal agent at the species level, but revealed its affinities to other Peronospora species parasitic on the Ranunculales and Saxifragaceae. To our knowledge, this is the first occurrence of a downy mildew affecting a species of Aquilegia. Already, about 1xa0year after its confirmed first occurrence in the UK and 3xa0years after reported symptoms, a huge impact on infested gardens and nurseries has occurred. As oospore production has been observed and the pathogen can grow systemically, rendering seed transmission likely, this pathogen should be classified as a high risk pathogen for Aquilegia. Appropriate quarantine measures should be taken to restrict the pathogen from spreading.

First report of tatsoi downy mildew caused by Hyaloperonospora brassicae in Korea.

Tatsoi (Brassica narinosa L.H. Bailey), also called spinach mustard or spoon mustard, is cultivated for edible greens in Asia. In Korea, this plant has recently become popular as a sprout vegetable that is grown to harvestable size in 5 to 6 days. During April 2012, tatsoi seedlings showing typical symptoms of downy mildew were found in plastic greenhouses in Pyeongtaek City of Korea. Infection resulted in chlorotic areas on the leaves with a white mildew developing on the abaxial surface, and finally led to necrosis of the lesions. Affected sprouts were unmarketable and abandoned without harvesting. A sample was deposited in the Korea University herbarium (Accession No. KUS-F26445). Microscopic examination of fresh material was performed under a light microscope. Conidiophores emerging from stomata were hyaline, 270 to 550 × 10 to 25 μm, straight, and monopodially branched in six to eight orders. Ultimate branchlets were mostly in pairs, flexuous, and 15 to 25 μm long. Conidia were hyaline, subglobose, and 20.5 to 26.5 × 19.5 to 24.5 μm with a length/width ratio of 1.05 to 1.20. These characteristics unequivocally indicate the genus Hyaloperonospora (1). Previously H. parasitica (formerly under Peronospora) has been considered a causal agent of downy mildew on tatsoi (2,4), but the present Korean accession is morphologically distinct from the former species by possessing subglobose conidia with a low length/width ratio. To confirm this morphological difference, amplification and sequencing of the internal transcribed spacer (ITS) region of rDNA of the Korean specimen were performed using procedures outlined by Göker et al. (3). The resulting 874-bp sequence of the region was deposited in GenBank (Accession No. JX401551). A comparison with the ITS sequences available in the GenBank database revealed that it was identical to Hyaloperonospora brassicae found on Brassica oleracea var. italica (EU137726), and showed only one base pair substitution compared to pathogens from B. rapa ssp. pekinensis (JF975613) and B. napus spp. napus (EU049248), but is significantly different from H. parasitica on Capsella bursa-pastoris (AY210988) with a base-pair dissimilarity of about 13%. Therefore, the pathogen found in Korea was confirmed to be H. brassicae. Pathogenicity was demonstrated by shaking diseased leaves onto the leaves of healthy tatsoi seedlings, incubating the plants in a dew chamber at 20°C for 24 h, and then maintaining them in a greenhouse (22 to 26°C). After 3 days, inoculated plants developed downy mildew symptoms from which identical fungi were observed, thus fulfilling Kochs postulates. Control plants treated with sterile water did not develop any symptoms or signs of downy mildew. This is the first report of a downy mildew on tatsoi in Korea, although it has been found in China (2) and Japan (4). To our knowledge, there is no record of tatsoi downy mildew outside East Asia (2,4). References: (1) O. Constantinescu and J. Fatehi. Nova Hedwigia 74:291, 2002. (2) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ July 18, 2012. (3) M. Göker et al. Mycol. Res. 113:308, 2009. (4) M. Satou et al. Annu. Rep. Soc. Plant Protect. N. Jpn. 50:62, 1999.

Phylogenomics of Bartheletia paradoxa reveals its basal position in Agaricomycotina and that the early evolutionary history of basidiomycetes was rapid and probably not strictly bifurcating

The higher level phylogeny of fungi has been addressed in previous studies, but for those analyses, either taxon sampling or gene sampling was low, or some basal lineages important for the inference of basidiomycete phylogeny were lacking. Here, a phylogenomic analysis based on highly conserved genes and including the enigmatic species Bartheletia paradoxa from Ginkgo biloba is presented. While phylogenetic analyses including also less conserved parts of core eukaryotic genes yielded a basal position for the extremophile genus Wallemia with low support, an exclusion of highly variable parts of these genes suggested Bartheletia paradoxa as the most basal member of the Agaricomycotina, but again with low support. Network analyses suggest a network-like evolution at the base of the Basidiomycota, supported by phylogenies based on single genes and gene clusters with shared topology. When further removing noise by removing poorly resolving genes, strong but not maximum support was obtained for Bartheletia paradoxa being the sister lineage to all other Agaricomycotina. We speculate that the lack of support for the early splits in Agaricomycotina and Basidiomycota can probably be explained by rapid radiation, linked to major evolutionary developments, such as, in the case of Basidiomycota, the advent of basidia in the last common ancestor.

Confirmation of Peronospora agrimoniae as a distinct species

Leaves with typical symptoms of downy mildew were found on common agrimony in the Czech Republic in 2014 and 2015 and at several locations in Germany from 2010 to 2014. The causal agent of downy mildew of agrimony was often reported as Peronospora agrimoniae, but sometimes also as P. sparsa. Morphological characteristics of the pathogens found in both countries are in the range of previous works for P. agrimoniae, but also other downy mildews parasitic on Rosaceae, rendering their discrimination based on published observations difficult. For molecular identification sequencing of several loci (nrITS rDNA, cox1 and cox2) was performed. Phylogenetic analyses based on nrITS rDNA clearly separated P. agrimoniae from other Peronospora species infecting Rosaceae. Thus, considering P. agrimoniae as separate species seems justified. Two German specimens were identical to two Czech samples in both nrITS rDNA and cox1 mtDNA sequences, but differed in a single nucleotide substitution in cox2 region. To our knowledge, this is the first verified record of P. agrimoniae on common agrimony in the Czech Republic.

The presumably North American species Plasmopara wilsonii is present in Germany on the ornamental plant Geranium phaeum

Geranium phaeum is a popular ornamental plant in Europe. Downy mildew disease caused by Plasmopara was found on this plant once 50xa0years ago in Poland, but at that time attributed to the native species, Plasmopara pusilla. Here we report the recurrence of downy mildew disease on G. phaeum in Germany. Molecular phylogenetic analysis and morphological investigations revealed that the causal agent of the new outbreak is P. wilsonii, but not P. pusilla. Plasmopara wilsonii hadxa0previously been found only in the USA and Korea on various hosts of Geranium. The presence of P. wilsonii on G. phaeum in Europe should be closely monitored, as downy mildew caused by this species probably has the potential to become an important emerging disease on its ornamental host, especially, as also the native G. molle is able to sustain the pathogen.

First Report of White Blister Rust Caused by Albugo candida on Wasabi in Korea

Wasabi (Wasabia japonica (Miq.) Matsum.), a member of the Brassicaceae family, is a commercially important crop in East Asian countries such as China, Japan, Korea, and Taiwan. In Korea, wasabi is under commercial development since it has become popular as a condiment due to its strong pungent constituents. In May 2013, wasabi plants showing typical symptoms of white blister rust disease were first observed in plastic greenhouses in Taebaek City, Korea. Leaves of infected plants had whitish sori on the lower surfaces and chlorotic blotches on the corresponding upper leaf surfaces. Later, sori changed to creamy to light tan with necrosis of leaf lesion. New infections might occur anytime during the growing season. A representative sample was deposited in the Korea University Herbarium (KUS-F27596). Microscopic examination of fresh materials was performed under a light microscope. The grouped sporangiophores were hyaline, clavate or cylindric, and measured 20 to 35 × 10 to 14 μm. The sporangia were arranged in basipetal chains, hyaline, globose to subglobose, with uniform wall thickness and measured 16 to 21 × 13 to 18 μm. The primary sporangia were morphologically similar to the secondary sporangia, although the former exhibited a slightly thicker wall than the latter. No resting organs were observed. Previously, the white blister rust pathogen on wasabi has been considered either Albugo candida or A. wasabiae, although the latter name is often considered a synonym of A. candida. Based on the morphological characteristics and the specific host plant, the causal agent of this disease was identified as A. candida (2). To confirm this morphological identification, genomic DNA was extracted from infected plant tissue, and the amplification and sequencing of the internal transcribed spacer (ITS) region of rDNA of the Korean specimen were performed using procedures outlined by Choi et al. (1), with oomycete-specific primer set, DC6 and LR0. The resulting 835-bp sequence of the region was deposited in GenBank (Accession No. KF887494). Since this was the first ITS sequence submitted for A. candida on wasabi, comparable data were not available. A comparison with the ITS sequences available in the GenBank database revealed that it is identical to A. candida found on Capsella bursa-pastoris (AF271231), and shows a high similarity of 99% with many A. candida sequences originating from other brassicaceous plants. Therefore, the pathogen found in Korea was confirmed to be A. candida. In Korea, it has been reported that A. candida attacks Brassica juncea, B. campestris subsp. penikensis, and B. napus (3), but to our knowledge this is the first record of A. candida on wasabi (4). The white blister rust caused by A. candida is one of the most devastating diseases of wasabi in Japan and Taiwan where the crop is widely cultivated. On the other hand, in the United States, Canada, and New Zealand, where wasabi is a new crop on a commercial scale, there is no record of this disease. These facts taken together suggest that wasabi white blister rust be not only currently spreading in East Asia, but it also poses a new and serious threat to production of this crop in countries in which it is currently absent. References: (1) Y. J. Choi et al. Mol. Phylogenet. Evol. 40:400, 2006. (2) Y. J. Choi et al. Fungal Divers. 27:11, 2007. (3) Y. J. Choi et al. Plant Pathol. J. 27: 192, 2011. (4) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., Online publication, ARS, USDA, Retrieved November 15, 2013.