Comparison of ITS Sequences from UK and North American Sugar-Beet Powdery Mildews and the Designation of Erysiphe Betae

mycological research 111 (2007) 204–212 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/mycres Comparison of ITS sequences from UK and North American sugar-beet powdery mildews and the designation of Erysiphe betae Sally A. FRANCISa, Brett C. RODENa, Michael J. ADAMSb, John WEILANDc, Michael J. C. ASHERa,* aBroom’s Barn Research Station, Higham, Bury St Edmunds, Suffolk IP28 6NP, UK bRothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK cUSDA Red River Valley Agricultural Research Service, Northern Crop Science Laboratory, Fargo, North Dakota 58105-5677, USA article info abstract Article history: Powdery mildew of sugar beet, a disease of major economic significance, was first Received 23 August 2005 described at the beginning of the 20th century, and since then there has been some confu- Received in revised form sion over the correct taxonomic identity of the causal agent. In Europe, the fungus was 28 August 2006 initially classified as the novel species Microsphaera betae, later re-named Erysiphe betae, Accepted 7 October 2006 whilst in America it was identified as E. polygoni, despite sugar-beet isolates from both re- Corresponding Editor: gions having a host range restricted to Beta species. It is possible that more than one fungus Derek T. Mitchell causes the disease, as published descriptions of conidiogenesis have differed. In this study, isolates of the fungus collected from sugar beet in the UK and USA were investigated for Keywords: polymorphisms in the rDNA ITS region to determine if the same species caused the disease Beta spp. in both countries, whether there was any justification for the retention of the name E. poly- Erysiphales goni in the USA, and to search for evidence of a second species infecting sugar beet. From Molecular systematics a total of 18 isolates examined, 23 ITS sequences were obtained. Fifteen of these, which Plant pathology included the UK and USA isolates, were identical and the remainder had single-base sub- Powdery mildews stitutions, indicating that the fungi were conspecific. Dendrogram analysis of Erysiphales ITS regions revealed that the UK and North American isolates were more closely related to E. heraclei than to E. polygoni. It is proposed that the species name Erysiphe betae be used for the powdery mildew fungus that infects sugar beet. No evidence was found in this study for a second sugar-beet powdery mildew species. ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction some countries, the fungus was only present in its anamorph stage at the time it was described. Despite a century of study, Infection of sugar beet (Beta vulgaris subsp. vulgaris, Amaran- the nomenclature of sugar-beet powdery mildew is still not thaceae, formerly Chenopodiaceae) by powdery mildew is fully standardised in the literature. economically significant for growers worldwide and can cause The disease was first described in Europe during the early sugar yield losses of up to 30 % (Francis 2002). Classifying the 20th century (Van˜ ha 1903) and it steadily spread through the causal agent of this disease has been difficult because, in continent, being recorded in the UK in 1935 (Anon. 1936), * Corresponding author. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2006.10.010 Sugar-beet powdery mildew ITS sequences 205 then reaching the USA two years later (Yarwood 1937). Van˜ ha long chains of conidia (catenate conidiogenesis). This is con- (1903) named the causal fungus Microsphaera betae based on tradictory to the short chains or single conidia reported chasmothecium (i.e. cleistothecium) appendage morphology elsewhere (Weltzien 1963; Drandarevski 1969; Mukhopadhyay and other characteristics. However, in the USA, sugar-beet & Russell 1979; Francis 2002). It has also been suggested that powdery mildew was initially identified as a form of Erysiphe the single case of the disease observed in the 1930s in the polygoni based on conidiophore morphology, in the absence USA might have been due to a different species than that of any chasmothecial samples (Yarwood 1937). Weltzien which caused an epiphytotic there in 1974 (Weltzien 1978). (1963) re-classified European isolates as a new species, E. betae, In the case of tomato powdery mildew, descriptions of coni- after an extensive biometrical study using a large number of diogenesis were contradictory and this led to the discovery chasmothecial samples. In the USA, powdery mildew that two species, Oidium lycopersici and O. neolycopersici, acted re-appeared as a major problem on sugar beet in the 1970s, as causal agents (Kiss et al. 2001). If more than one powdery this time producing chasmothecia. The fungus was again clas- mildew species infects sugar beet, this would create complica- sified as E. polygoni by Coyier et al. (1975) as the size of the chas- tions in powdery mildew resistance breeding programmes mothecia fell within the range previously reported for (Francis 2002; Francis & Luterbacher 2003). Arabidopsis can be E. polygoni and they did not consider that host range was infected by two powdery mildews, E. cruciferarum and Golovi- a sufficient criterion for speciation. nomyces cichoracearum, and large-scale testing has revealed The value of host-range information in classifying that accessions contain resistance to both, neither, or either powdery mildew fungi has been controversial (Adam et al. one of the fungi (Adam et al. 1999; Vogel & Somerville 2002). 1999), although it can be used for taxonomic purposes (Braun Investigation of ITS polymorphisms is a powerful technique 1995; Braun et al. 2002). Van˜ ha (1903) used host-range informa- for resolving whether more than one powdery mildew is pres- tion in his original description of sugar-beet powdery mildew. ent. In a recent study, Cunnington et al. (2004) used ITS Weltzien (1963) noted that E. polygoni had not previously been sequence data to discover a previously-unknown powdery reported on any member(s) of the Chenopodiaceae, so argued mildew taxon on Delphinium, and Takamatsu et al. (2002) that it was unlikely to be the cause of sugar-beet powdery were able to distinguish E. glycines and E. diffusa, two species mildew, although he did not perform any host range experi- that can simultaneously infect soybean but have virtually ments with the sugar-beet isolates. Positive proof of the nar- identical anamorphs. row host-range of sugar-beet powdery mildew (from Europe) In this study, isolates have been taken as either cellulose was gained when the fungus was test-inoculated onto 52 acetate peels from the surfaces of sugar-beet leaves, or as crop, weed and wild halophytic species (Drandarevski 1969). spores vacuumed or rinsed from leaf surfaces, and are thus The results showed that the beet powdery mildew fungus parts of powdery mildew colonies. Sequences of the ITS was restricted to Beta species. Similarly, in the USA, test inoc- regions of UK and North American sugar-beet powdery mil- ulations of powdery mildew isolates from sugar beet onto 33 dew isolates were determined, compared with each other, different plant species showed that the fungus could only in- and with other Erysiphales ITSs deposited in the sequence fect, and sporulate, on sugar beet (Ruppel & Tomasovic 1977). databases (including sequences deposited since Saenz & In addition, an E. polygoni isolate collected from Rumex crispus Taylor’s 1999 study), to determine: (1) whether there were growing in a sugar-beet field could not infect sugar beet. any differences between the UK and North American isolates; Further studies have since identified Chenopodium ambro- (2) whether the name E. betae or E. polygoni was most suitable soides, C. anthelminticum and C. botrys in Asia (Braun 1987; for the fungus; and (3) whether there was any evidence for Otani 1988; Nomura 1997) and Celosia sp. (Amaranthaceae)in a second, cryptic, powdery mildew fungus on sugar beet in Germany (Braun 1998) as hosts of E. betae, based on studies either region. of chasmothecium morphology and other characters. As well as morphological and host-range data, a third char- acteristic useful in the taxonomic classification of obligate Materials and methods biotrophic plant pathogens such as powdery mildew fungi is DNA sequence variation. Many studies have successfully Collection of isolates and DNA extraction used rDNA ITS sequence polymorphisms (White et al. 1990) to differentiate Erysiphales species and study their phylogeny Sugar-beet leaves that were heavily infected with powdery (Takamatsu et al. 1998; Takamatsu et al. 1999; Saenz & Taylor mildew, but not senescent or exhibiting symptoms of other 1999; Mori et al. 2000; Kiss et al. 2002, 2005; Cunnington et al. pests or diseases, were used as sources of the fungus. For 2003). PCR amplification of the ITS region is a technique admi- the UK isolates, mycelium was peeled from leaf surfaces by rably suited to obligate pathogens because of its success painting the leaves with a ‘glue’ of cellulose acetate dissolved despite the often very small amounts of template DNA that in acetone, which was then stripped from the leaves, along are available. Saenz & Taylor (1999) included, amongst many with the mycelium, after the acetone had evaporated (White- other species, E. betae and E. polygoni in their dendrogram house et al. 1982; Francis 1996). This process avoids contami- analyses and revealed the two species to be closely related, nating the fungal sample with host plant DNA. The acetate but not necessarily conspecific. ‘peels’ were immediately frozen in liquid nitrogen, ground A further line of investigation that needs developing is with DNase-free sand in microcentrifuge tubes, and DNA whether more than one powdery mildew fungus can infect was extracted from the resultant powder using a Nucleon sugar beet. In details of UK isolates given by Hull (1949, Phytopure kit (GE Healthcare, Chalfont St Giles, UK).

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