Molecular evidence in support of recent ... - Wiley Online Library

Plant Pathology (2008) 57, 243–250

Doi: 10.1111/j.1365-3059.2007.01775.x

Molecular evidence in support of recent migration of a powdery mildew fungus on Syringa spp. into Europe from East Asia Blackwell Publishing Ltd

Y. Sekoa, A. Bolayb, L. Kissc, V. Helutad, B. Grigaliunaitee and S. Takamatsua* a Department of Bioresources, Graduate School, Mie University, 1577 Kurima-Machiya, Tsu 514-8507, Japan; bConservatoire et Jardin Botaniques de la Ville de Genève, CH-1292 Chambésy, Switzerland; cPlant Protection Institute of the Hungarian Academy of Sciences, P.O.B. 102, H-1525 Budapest, Hungary; dInstitute of Botany of the National Academy of Sciences of Ukraine, 2 Tereshchenkivska St., Kiev 01601, Ukraine; and eInstitute of Botany, Laboratory of Phytopathogenic Microorganisms, LT-8406 Vilnius, Lithuania

A preliminary analysis demonstrated that the powdery mildew fungus infecting Syringa spp. (lilacs) in different parts of the world is divided into two groups (S-type and K-type) based on the nucleotide sequences of the rDNA ITS regions. In spite of the marked genetic differences (only c. 94% similarity between ITS types), fungi belonging to these two ITS groups are difficult to distinguish based on morphological characteristics. To determine their geographical distribution, ITS haplotypes were determined for a total of 139 powdery mildew specimens collected in Asia, Europe and North and South America between 1977 and 2005. Curiously, until 1990, only the S-type was found in Europe, whilst the K-type prevailed in East Asia. The first European specimen belonging to the K-type was collected in Ukraine in 1991. Other European K-type samples were collected in Lithuania and Switzerland in 2000, and in other countries after 2002. The incidence of the S-type decreased rapidly in Europe after the 1990s. This result strongly suggests that the K-type was introduced to Europe from East Asia in the 1990s and expanded from Eastern Europe westward, replacing the S-type present on the European lilacs. The K-type produces abundant chasmothecia (sexual fruiting bodies), whereas chasmothecia on the S-type are rare in Europe and East Asia. It is likely that the recent abundant production of chasmothecia on Syringa spp. in Central Europe is explained by the migration of the K-type to Europe. Keywords: Erysiphales, ITS region, lilac, PCR-RFLP, phylogeography, rDNA sequence

Introduction Global trade and other international human activities have inadvertently resulted in the introduction and spread of a large number of plant pathogenic microorganisms into new environments (e.g. Britton, 2004). Some of these have already caused serious problems in the newly occupied areas, such as potato late blight, coffee rust, grapevine powdery mildew, Fusarium wilt of banana, and chestnut blight (Bulit & Lafon, 1978; Su et al., 1986; Schumann, 1993). Recently, a number of powdery mildew species were reported for the first time in Europe; these included Erysiphe azaleae infecting rhododendron (Rhododendron spp.) (Ing, 2000; Inman et al., 2000; Heluta et al., 2004), E. flexuosa infecting horse chestnut (Aesculus hippocastanum) (Ale-Agha et al., 2000; Heluta & Voytyuk, 2004; Kiss et al., 2004), E. elevata on Catalpa

*E-mail: [email protected] Accepted 12 August 2007

© 2007 The Authors Journal compilation © 2007 BSPP

bignonioides (Ale-Agha et al., 2004; Vajna et al., 2004; Cook et al., 2006) and E. symphoricarpi on snowberry (Symphoricarpos albus) (Kiss et al., 2002; Szentiványi et al., 2004). All these fungi are thought to have been introduced from North America to Europe (Kiss, 2005). Species of Syringa, the lilacs, are common host plants of the powdery mildew fungi. Lilacs are known for their beautiful and fragrant flowers and are widely cultivated as ornamentals in many parts of the world. Twenty-two to 28 species of Syringa are distributed in southern Europe and East Asia (McKelvey, 1928; Fiala, 1988; Chang et al., 1996). One Phyllactinia species and two Erysiphe (section Microsphaera) species were reported on Syringa (Wheeler, 1978; Amano, 1986; Braun, 1987). Erysiphe syringae was described in 1834 based on a specimen collected in North America, and is also known to occur in Europe, Siberia and Australia. Erysiphe syringae-japonicae was described by Braun (1982) as a new species based on a specimen collected in Japan, and has been considered to be endemic to East Asia. Braun (1982) distinguished E. syringae-japonicae from E. syringae by pigmentation of appendages and number of ascospores in an ascus.

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It is thought that E. syringae was introduced to Europe in the 19th century from North America, its putative geographic origin (Braun, 1987), and became widely distributed in Europe by the middle of the 20th century (Blumer, 1951). Formation of ascomata (fruiting bodies now known as chasmothecia) of E. syringae is common in North America, while it has been very rare in Europe (Blumer, 1951; Braun, 1987). However, since 1998, a striking increase has been observed in the number of chasmothecia produced on lilacs in Germany and Switzerland. The causal fungus was identified as E. syringaejaponicae (Braun, 1998; Bolay, 2005). A possible and reasonable explanation for this phenomenon is that E. syringae-japonicae has been newly introduced to Europe from East Asia. Bolay (2005) examined two North American E. syringae specimens, collected in California in 1900 and in New York in 1955, and further specimens collected more recently in North America. All these American specimens differed from E. syringae, based on morphological patterns, and could clearly be identified as E. syringae-japonicae. However, only E. syringae is known to occur in North America (Braun, 1987). Thus, Braun (1998) suggested that the identity of E. syringae in both North America and Europe needs to be re-examined. Bolay (2005) concluded that E. syringae-japonicae is conspecific to E. syringae based on morphology. However, it is also possible that E. syringae-japonicae has also been introduced in North America and replaced E. syringae during the past decades (U. Braun, Martin-LutherUniversität, D-06 099 Haale (Saale), Germany, personal communication). If E. syringae and E. syringae-japonicae are conspecific, the earlier speculation that the recent increase in chasmothecia on lilac powdery mildew in Europe is the result of the introduction and spread of E. syringae-japonicae in these countries needs to be reconsidered. Clearly, molecular methods, such as DNA analyses, are needed to test this point and also to precisely identify the species involved. In this study, DNA sequencing and PCR-RFLP analysis of the rDNA internal transcribed spacer (ITS) region were conducted on a number of powdery mildew specimens infecting lilacs, collected between 1977 and 2005 from different parts of the world. The aims of the study were to reveal (i) the identity and genetic variation of powdery mildew fungi of the genus Erysiphe parasitic to lilacs, (ii) the geographic distributions of various ITS types of the lilac mildews, and (iii) the explanation of the considerable increase in numbers of chasmothecia on lilac in Europe since 1998.

Materials and methods

PCR and DNA sequencing The ITS regions, including the 3′ end of the 18S (small subunit) rRNA gene, the 5·8S rRNA gene and the 5′ end of the 28S (large subunit) rRNA gene, were amplified by the polymerase chain reaction (PCR) with the primer pair ITS5 (White et al., 1990) and P3 (Kusaba & Tsuge, 1995) for the first amplification. The first PCR products were used for the templates of the second PCR using the nested primer set ITS5/ITS4, ITS1/ITS4 (White et al., 1990) or PM7[RYYGACCCTCC(C)ACCCGTGY]/ITS4. PCR reactions were conducted with TaKaRa Taq DNA polymerase in a thermal cycler TP-400 (TaKaRa) under the following conditions: an initial denaturing step for 2 min at 95°C, then 30 cycles each consisting of 30 s at 95°C, 30 s at 52°C for annealing and 30 s at 72°C for extension, and a final extension cycle of 7 min at 72°C. A negative control that lacked template DNA was included for each set of reactions. The PCR amplicons were separated by electrophoresis on 1·5% agarose gels in TAE buffer. The desired band was visualized under a long-wavelength ultraviolet light and cut out of the gel. Purification of the DNA fragment was performed utilizing the JETSORB kit (GENOMED), following the manufacturer’s protocol. Both strands of the amplicons were sequenced using the primers ITS5, ITS4, ITS2 (White et al., 1990) and T4 (Hirata & Takamatsu, 1996). The sequence reactions were conducted using the CEQ Dye Terminator Cycle Sequencing Kit (Beckman Coulter) according to the manufacturer’s instructions, and run on a DNA sequencer CEQ2000XL (Beckman Coulter).

Phylogenetic analysis The sequences were initially aligned using the clustal x package (Thompson et al., 1997). Alignment was then visually refined with a word processing program, using colour-coded nucleotides, and ambiguously aligned sites were removed from the dataset in the following analyses. The alignments were deposited in TreeBASE (http:// www.treebase.org/) under the accession number S1895. Phylogenetic trees were obtained from the data using the maximum-parsimony (MP) method in paup* 4·0 (Swofford, 2001). MP analyses were performed with the heuristic search option using the ‘tree-bisection-reconstruction’ (TBR) algorithm with 100 random sequence additions to find the global optimum tree. All sites were treated as unordered and unweighted, with gaps treated as missing data. The strength of the internal branches of the resulting trees was tested with bootstrap analyses using 1000 replications (Felsenstein, 1985).

Sample collection A total of 139 powdery mildew specimens found on Syringa spp. in Asia, Europe and North and South America were collected in the field or obtained from existing collections. Whole-cell DNA was extracted from chasmothecia or mycelia by the chelex method (Walsh et al., 1991; Hirata & Takamatsu, 1996).

Restriction enzyme digestions The amplified PM7/ITS4 fragment (580bp) was digested with HindIII (Toyobo) or BcnI (Fermentas) according to the manufacturer’s recommendations. Digestion mixtures were prepared as 10 U enzyme, 1·5 μL enzyme buffer (supplied by the manufacturer), 7·5 μL sterile distilled Plant Pathology (2008) 57, 243–250

Migration of lilac powdery mildew in Europe

water and 5 μL PCR product per tube. Digestions were run for 2 h at 37°C. Restricted DNA was then analysed on 2% agarose gels in TBE buffer.

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deposited under the accession numbers AB295458 (K), AB295460 (S-1) and AB295462 (S-2).

Phylogenetic analysis Scanning electron microscopic (SEM) observations Fresh leaves with chasmothecia were cut into small pieces with a razor blade then treated by the modified tannic acid fixation method (Kunoh et al., 1977). The specimens were fixed with 2% unbuffered glutaraldehyde containing 0·2% tannic acid at room temperature (RT) for 12 h, followed by further fixation in 2% unbuffered glutaraldehyde containing 2% tannic acid at RT for 12 h. After being washed in deionized water for 2 h, the specimens were treated in 1% unbuffered aqueous OsO4 at RT for 12 h and washed in deionized water for 1 h. They were dehydrated in a graded ethanol series, then in a graded ethanol- iso -amyl acetate series and finally placed into 100% iso-amyl acetate. They were then dried in a critical-point dryer and coated with gold using an ionsputter (model E-1010, Hitachi). Specimens were observed with a SEM (model S-4000, Hitachi) at 20 kV accelerating voltage.

Production of chasmothecia The incidence of chasmothecium formation by isolates was observed under a stereomicroscope (Olympus SZ-11) and evaluated as follows: – no chasmothecia observed, + less than 50 chasmothecia per leaf, ++ intermediate between 50 and 200, and +++ more than 200 chasmothecia per leaf.

Results Sequence analysis DNA sequences of rDNA ITS regions were determined for a total of 34 powdery mildew specimens from Syringa spp. These sequences were divided into two groups. The sequences of the first group were 99·8–100% identical to the DNA database sequence AB015920, the ITS sequence of E. syringae. The sequences of the other group were 99·8% identical to the sequence AB015917, the ITS sequence of E. katumotoi on Ligustrum obtusifolium. Because E. katumotoi is considered to be a synonym of E. ligustri (Shin, 2000), this species is hereafter referred to as E. ligustri. The first ITS group was designated ‘S’ and the second group ‘K’. Sequence similarities were only 93·9–94·1% between the two ITS types. ITS regions including 5·8S rDNA were 557 bp long in the S-type and 553 bp in the K-type. The S-type was further divided into two haplotypes, whereas the K-type consisted of a single haplotype. Nucleotide substitutions were only one base between the two haplotypes of the S-type. The S-type was found in a total of 21 specimens. Out of these, 19 specimens represented haplotype S-1 and two of them belonged to haplotype S-2. The remaining 13 specimens were K-type. The representative sequences from each haplotype were Plant Pathology (2008) 57, 243–250

Three ITS sequences representing each haplotype were aligned with sequences of the sections Erysiphe and Microsphaera of the genus Erysiphe retrieved from the DNA Data Bank of Japan. The alignment data matrix consisted of 48 taxa and 605 characters, of which 47 characters were removed from the following analysis because of ambiguous alignment. Of the remaining 558 aligned sites, 178 (31·9%) characters were variable and 124 (22·2%) characters were informative for parsimony analysis. Erysiphe glycines was used as an outgroup taxon based on the report of Takamatsu et al. (1999). This parsimony analysis using paup* generated 181 equally parsimonious trees of 417 steps [consistency index (CI) = 0·5683, retention index (RI) = 0·7497, rescaled consistency index (RC) = 0·4261]. Tree topologies were almost consistent among the 181 trees, except for small branching orders of the terminal branches and branch length. One of the 181 trees with the highest log likelihood value is shown in Fig. 1. The two ITS types did not group together. The two S haplotypes formed a monophyletic group, whereas the K-type grouped with E. ligustri; in each of these cases bootstrap values were 99–100%.

Geographic distribution of ITS types PCR-RFLP analysis was used to identify the ITS types in a total of 105 specimens, in addition to the 34 specimens included in the sequence analysis. Based on the ITS sequence data, restriction enzymes HindIII and BcnI were chosen to distinguish the two ITS types. HindIII digested ITS regions of K-type into two fragments (420, 160bp), but did not digest S-type ITS (Fig. 2). BcnI digested K-type ITS into two fragments (530, 50bp) and S-type ITS into three fragments (370, 160, 50bp). Of the 139 specimens collected between 1977 and 2005, 33 were S-type and the remaining 106 were K-type. The incidences of ITS types from Syringa spp. were plotted on a world map over various collection periods (Fig. 3). Between 1977 and 1990, the S-type was detected from both Japan and Europe, while the K-type was detected only from Japan and the far east of Russia (Fig. 3a). The K-type was first found in Europe (Ukraine) in 1991, and in 2000–01 in Lithuania, Germany and Switzerland (Fig. 3b). All specimens collected in Europe between 2002 and 2005 were K-type, except for a specimen collected in Ukraine (Fig. 3c). Only S-type was detected in North and South America between 1977 and 2005.

Production of chasmothecia The production of chasmothecia was investigated in 131 powdery mildew specimens collected in autumn (of these, 28 belonged to the S-type and 103 to the K-type). Of the 28 S-type specimens, chasmothecia were observed only in

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Figure 1 Phylogenetic analysis based on rDNA ITS dataset for 48 taxa of the genus Erysiphe, including three representative sequences from powdery mildews on Syringa spp. The tree is a phylogram of the maximum likelihood tree among the 181 most parsimonious trees, with 417 steps, found using a heuristic search employing the random stepwise addition option of PAUP* 100 times, treating gaps as missing data. Horizontal branch lengths are proportional to the number of nucleotide substitutions inferred to have occurred along a particular branch of the tree. Maximum-parsimony bootstrap values > 50% are shown on branches.



Figure 2 PCR-RFLP analysis of the rDNA internal transcribed regions of the powdery mildew isolates from Syringa spp. HindIII digests ITS regions of K-type into two fragments, but did not digest S-type ITS. BcnI digests K-type ITS into two fragments (530, 50bp) and S-type ITS into three fragments (370, 160, 50bp). The shortest 50bp band is not shown.

nine (Fig. 4). Of these, six were collected in North America, a putative geographic origin of E. syringae. Chasmothecia were not found in S-types from Japan and South America, and were rare in specimens from Europe. On the other hand, chasmothecia were abundant in all 103 K-type specimens studied.

Discussion The K-type isolates from Syringa spp. were closely related to E. ligustri on Ligustrum spp. in phylogeny. However, there may be genetic segregation between the isolates from Syringa spp. and E. ligustri because the ITS sequence of Syringa isolates usually differed by at least one base from the sequence of Ligustrum isolates. SEM observation revealed that E. ligustri from Ligustrum spp. had chasmothecial appendages with a three-dimensional apical branching pattern (Fig. 5a) characteristic of the species (Shin, 2000). On the other hand, K-type isolates from Syringa spp. had appendages with a two-dimensional apical branching pattern (Fig. 5b), clearly differing from E. ligustri. K-type isolates from Syringa spp. were similar to E. syringae-japonicae in morphological characteristics. Moreover, although cross-inoculation tests between Syringa and Ligustrum are required, it is expected that these isolates would also differ in host range or at least in aggressiveness on the respective hosts. Therefore, although K-type isolates from Syringa spp. and E. ligustri are closely related to each other in phylogeny, they should be regarded as different species. According to Braun (1987), appendages of E. syringae are hyaline, or faintly pigmented at the base, and ascospore number is 3 – 6(7), mostly 4 –5. In contrast, appendages of E. syringae-japonicae are pigmented from the base to the upper half, or sometimes uniformly brown from the middle downward, and ascospore number is 6–8. In the present observations, pigmentation Plant Pathology (2008) 57, 243–250

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of appendages of S-types was somewhat paler than that of K-types, but in some S-type specimens appendages were pigmented from the base to the upper half, similar to E. syringae-japonicae. In addition, ascospore number in an ascus was 4–7 in S-types and 5–8 in K-types, indicating that both ITS types are closer to E. syringae-japonicae than to E. syringae in morphology. Thus, S-types could not be identified as E. syringae. More comprehensive morphological analyses are required for a taxonomic treatment of the powdery mildew fungi from lilacs belonging to the two distinct ITS types. The geographic distributions of the two ITS types strongly suggested that the K-type was newly introduced to Europe from East Asia in the 1990s and spread from Eastern Europe westward. One of the aims of the present study was to reveal the cause of the increase in chasmothecia on lilac in Central Europe since 1998 (Braun, 1998; Bolay, 2005). This could be the result of migration of the K-type into Europe. To address the question, the incidence of chasmothecia production by ITS types was investigated. The results clearly indicated that K-types produce abundant chasmothecia, while they are rare in S-types, except for the collections from North America (Fig. 4). This suggests that the recent abundant development of chasmothecia of lilac mildew is the result of the migration of K-type fungi to Europe from East Asia. It seems that based on the production of chasmothecia in the newly occupied regions, the invasive powdery mildews belong to two main groups. Some of these species, such as E. necator, E. arcuata and E. symphoricarpi, did not produce chasmothecia for many years/decades following their introduction to a new environment, while their sexual reproduction remained unchanged in their native areas (Kiss et al., 2002). In other invasive powdery mildew species, such as E. elevata and E. flexuosa, sexual reproduction was not affected by spread to a new continent (Ale-Agha et al., 2000; Vajna et al., 2004). In lilac powdery mildew, however, the recent increase in the production of chasmothecia is not the result of a change in the reproductive system of a single species. Apparently, it has happened because of a recent introduction of another, morphologically similar species that infects the same host plant species. Thus, this possibility should always be considered when chasmothecia suddenly appear on hosts where sexual reproduction was not previously observed. In this study, numerous specimens collected during the past 30 years were used to reveal the geographic distribution of the lilac powdery mildews. As the fungus introduced to Europe (K-type) is difficult to distinguish from the existing fungus (S-type) using morphological characteristics, its spread could only be reliably followed using molecular methods. This is the first molecular phylogeographical study in the Erysiphales.

Acknowledgements Since thanks are due to Drs M. Havrylenko (Bariloche, Argentina), Y. Nomura (Chiba, Japan), Y. Sato (Toyama,

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Figure 3 Geographic distributions of S-type (open circle) and K-type (closed circle) of the powdery mildew (Erysiphe spp.) isolates from Syringa spp. collected between 1977 and 2005.



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Japan), A. Schmidt (Lübeck, Germany) and S. Tanda (Tokyo, Japan) for kindly providing specimens, and also to the editor and the anonymous reviewers for the helpful comments and suggestions. This work was supported in part by Grants-in-Aid for Scientific Research (15405021) from the Japan Society for the Promotion of Science (JSPS).

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Figure 4 Incidence of chasmothecium production by S-type and Ktype of the powdery mildew (Erysiphe spp.) isolates from Syringa spp. – no chasmothecia observed; + less than 50 chasmothecia per leaf;++ intermediate between 50 and 200; +++ more than 200 chasmothecia per leaf.

Figure 5 Scanning electron microscopy of chasmothecia of Erysiphe ligustri on Ligustrum tschonoskii f. glabrecens (a) and K-type fungus on Syringa vulgaris (b). These two fungi were closely related in terms of rDNA ITS sequences (99·8% identity), but were distinguishable by the apical branching pattern of appendages: three-dimensional in E. ligustri and two-dimensional in the K-type.


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