Identification of the pathogen Podosphaera erigerontis-canadensis causing powdery mildew disease on dandelion (Taraxacum officinale) in US Arkansas state
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Powdery mildew was observed on common dandelion (Taraxacum officinale), collected in Fayetteville, AR, USA, and was identified using morphology and DNA sequences of ITS and 28S ribosomal RNA (rRNA) gene regions. Four primer pairs were used in this study: (1) the powdery mildew specific primer pair PMITS1/2 produced a 696 bp DNA fragment including full sequence of 563 bp DNA fragment amplified by ITS1/4 (KU563734); (2) CTW13/TW14 created a 279 bp DNA fragment including partial sequence of 28S rRNA gene (KU563735); (3) the 28S rRNA region primer pair NL1f/TW14 amplified a 899 bp DNA fragment (KX610821); and (4) the primer pair ITS1/4 amplified a 563 bp DNA fragment including partial sequence of 18S ribosomal RNA gene and complete sequence of ITS1, the 5.8S rRNA gene, ITS2, and partial sequence of the 28S rRNA gene. The similarity analysis by Blast and phylogenetic analysis indicated that the powdery mildew pathogen in dandelion collected from Arkansas was Podosphaera erigerontis-canadensis.
KeywordsDandelion Internal transcribed spacer (ITS) Podosphaera erigerontis-canadensis Powdery mildew Taraxacum officinale
Powdery mildew is one of the most common and widespread plant diseases (Ale-Agha et al. 2013). Most powdery mildew diseases are caused by the Erysiphaceae family which consists of 16 genera and approximately 900 species (Braun and Cook 2012). Erysiphe is the largest genus in Erysiphaceae (Takamatsu et al. 2015), followed by Golovinomyces and Podosphaera. Although powdery mildew disease is caused by different species and occurs on a wide range of plants, the symptoms of powdery mildew are similar: white to greyish spots, and powder-like cover on leaves, stems, and sometimes fruit. Powdery mildew usually affects young tissue, including young leaves. The white spots gradually spread to cover most of the leaves, and finally leaves turn yellow and dry out. Morphological criteria and host ranges are the classical methods to identify fungal pathogens. In recent decades, internal transcribed spacer (ITS) as a universal DNA barcode marker have been used for fungal diagnostics and phylogeny (Schoch et al. 2012). The study of biology and taxonomy of fungi is efficient ways to understand the species of powdery mildews in a geographic region (Khodaparast and Abbasi 2009). Molecular re-examination of Podosphaera fusca resulted in delineation of Podosphaera erigerontis-canadensis on T. officinale (Takamatsu et al. 2010) and morphologically described P.fusca on Conyza canadensis in France (Braun and Cook 2012), Taraxacum sp. in Iran (Sharifi et al. 2014) and Crepis sp. and Erigeron sp. in Sweden (Eriksson 2014). Prior to taxonomic revisions of powdery mildew species, P. erigerontis-canadensis was reported under the name Sphaerotheca erigerontis-canadensis, S. fuliginea, S. fusca, S. macularis and S. humuli on a range of hosts including T. officinale in the USA (Farr et al. 1989; Braun et al. 2013) and worldwide. However, there are no records of this fungus colonising T. officinale in Arkansas, the USA. Since 2014, the Vegetable Breeding program at the University of Arkansas has started a new research project on common dandelion, and the purpose is to develop common dandelion as a vegetable. Therefore, the present research aimed to investigate the powdery mildew species on dandelion.
DNA was extracted from mycelia and conidia using the CTAB protocol (Shi and Mmbaga 2006; Takamatsu et al. 2010). Polymerase chain reactions (PCRs) were performed using four pairs of primers to amplify ITS and 28S rDNA regions: (1) powdery mildew specific ITS primers PMITS1 (5′-tcggactggcccagggaga-3′)/PMITS2 (5′-tcactcgccgttactgaggt-3′) (Cunnington et al. 2003), (2) CTW13 (5′-cgtcttgaaacacggacc-3′)/TW14 (5′-gctatcctgagggaaacttc-3′) (source: http://nature.berkeley.edu/brunslab/tour/primers.html), (3) NL1f (5′-tgggtggtaaattccatcta-3′)/TW14) (White et al. 1990; source: http://nature.berkeley.edu/brunslab/tour/primers.html), and (4) universal ITS primers ITS1 (5′- tccgtaggtgaacctgcgg-3′)/ITS4 (5′-tcctccgcttattgatatgc-3′) (White et al. 1990). PCR products were purified by using OMEGA E.Z.N.A.® Cycle Pure Kit. The purified DNA products were sequenced from 5′ and 3′ directions by Eurofins Genomics Company, and consensus sequences were assembled and used for subsequent phylogenetic analyses.
For similarity analysis to other pathogens of powdery mildew, a seven nucleotide difference was found after comparison with the sequence of Podosphaera xanthii (GenBank: KM260741), and eight nucleotides were identified to be different from the sequence of Podosphaera balsaminae (GenBank: KR048106). Other 28S rDNA sequences of powdery mildew fungi in GenBank including Podosphaera xanthii (GenBank: AB462760), Podosphaera balsaminae (Genbank: AB462789), Podosphaera dicilipterae (GenBank: AB462795), Podosphaera euphorbiae-hirtae (GenBank: AB462770), Podosphaera cayratiae (GenBank: AB462765), and Podosphaera astericola (GenBank: AB462779), found that the DNA sequence amplified by the primer NL1f/TW14 from the powdery mildew fungus identified in Arkansas, USA had one nucleotide difference, further indicating that the P. erigerontis-canadensis was a distinct species causing powdery mildew on dandelion in Arkansas.
In this study, a total of 39 ITS and 19 28S sequences of powdery mildew pathogens were used for phylogenetic analysis by using MEGA 6 (Tamura et al. 2013) aligned by MUSCLE method. The results, shown in Figs. 3 and 4, indicated that there were no phylogenetic difference between the fungal pathogen found in Fayetteville, Arkansas, United States and in Argentina by Takamatsu (GenBank: AB525916).
This species has not been documented in Arkansas, the USA. Other powdery mildew species reported on dandelion in the USA were Sphaerotheca humuli/S. humuli var. fuliginea (Plant Pathology Database, University of Massachusetts Amherst- https://urldefense.proofpoint.com/v2/url?u = https-3A__www.bio.umass.edu_biology_research_herbarium_plantpathology_archive.php-3Fgenus-3DSphaerotheca&d = DwIGaQ&c = 7ypwAowFJ8v-mw8AB-SdSueVQgSDL4HiiSaLK01W8HA&r = f2dIVbIi-OnCTqImm6VdMg&m = _tlJulcwLz6_4ztydi15mPl1WbzBYxlrRsB2gT2hL_E&s = ruj7bhFfnyGgp4kcAsYqald1WXCywgddunTSLZ5P6tM&e=), Golovinomyces cichoracearum and Phyllactinia guttata (Farr et al. 1989). Identification of these species was based on morphological markers that are distinct from the morphological markers associated with P. erigerontis-canadensis. Oidium subgenus Fibroidium was also reported on dandelion in Japan (Satou et al. 2012) and its identification was based on the sequencing of the ITS region. Our DNA sequencing data and phylogenetic analysis indicate that this is the first report of P. erigerontis-canadensis on dandelion in Arkansas, the USA.
This work is supported, in part, by the USDA National Institute of Food and Agriculture Hatch project accession number 1002423.
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