Tünde Jankovics

Ampelomyces mycoparasites from apple powdery mildew identified as a distinct group based on single-stranded conformation polymorphism analysis of the rDNA ITS region

Pycnidial fungi belonging to the genus Ampelomyces are the most common natural antagonists of powdery mildews worldwide. During a study of the interactions between apple powdery mildew (Podosphaera leucotricha) and Ampelomyces mycoparasites, 52 new Ampelomyces isolates were obtained from P. leucotricha and, in addition, 13 new isolates from other species of the Erysiphaceae in four European countries. Their genetic diversity was screened using single-stranded conformation polymorphism (SSCP) analysis of the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA). For comparison, 24 isolates obtained from genetic resource collections or other sources were included in this study. Based on the ITS-SSCP patterns, the isolates were placed in eight groups. The isolates belonged to two types based on their growth in culture. The faster-growing and the slower-growing isolates were included in different SSCP groups. A phylogenetic analysis of the ITS sequences of representatives of these groups confirmed the results obtained with the SSCP method, and showed that the faster-growing isolates do not belong to Ampelomyces as suggested by earlier studies. All the isolates from P. leucotricha fell into a distinct SSCP group of genetically homogeneous isolates. This suggests that Ampelomyces mycoparasites which occur in apple powdery mildew are slightly different from the other Ampelomyces groups which contain mycoparasites from various powdery mildew species. This may be because the main growth period of Ampelomyces mycoparasites in apple powdery mildew is isolated in time from that of Ampelomyces isolates that occur in other species of the Erysiphaceae. P. leucotricha starts its life-cycle early in the season, usually in March-April, while most powdery mildews are active in the same environments only late in the year.

Oidium neolycopersici: Intraspecific variability inferred from amplified fragment length polymorphism analysis and relationship with closely related powdery mildew fungi infecting various plant species

Previous works indicated a considerable variation in the pathogenicity, virulence, and host range of Oidium neolycopersici isolates causing tomato powdery mildew epidemics in many parts of the world. In this study, rDNA internal transcribed spacer (ITS) sequences, and amplified fragment length polymorphism (AFLP) patterns were analyzed in 17 O. neolycopersici samples collected in Europe, North America, and Japan, including those which overcame some of the tomato major resistance genes. The ITS sequences were identical in all 10 samples tested and were also identical to ITS sequences of eight previously studied O. neolycopersici specimens. The AFLP analysis revealed a high genetic diversity in O. neolycopersici and indicated that all 17 samples represented different genotypes. This might suggest the existence of either a yet unrevealed sexual reproduction or other genetic mechanisms that maintain a high genetic variability in O. neolycopersici. No clear correlation was found between the virulence and the AFLP patterns of the O. neolycopersici isolates studied. The relationship between O. neolycopersici and powdery mildew anamorphs infecting Aquilegia vulgaris, Chelidonium majus, Passiflora caerulea, and Sedum alboroseum was also investigated. These anamorphs are morphologically indistinguishable from and phylogenetically closely related to O. neolycopersici. The cross-inoculation tests and the analyses of ITS sequences and AFLP patterns jointly indicated that the powdery mildew anamorphs collected from the above mentioned plant species all represent distinct, but closely related species according to the phylogenetic species recognition. All these species were pathogenic only to their original host plant species, except O. neolycopersici which infected S. alboroseum, tobacco, petunia, and Arabidopsis thaliana, in addition to tomato, in cross-inoculation tests. This is the first genome-wide study that investigates the relationships among powdery mildews that are closely related based on ITS sequences and morphology. The results indicate that morphologically indistinguishable powdery mildews that differed in only one to five single nucleotide positions in their ITS region are to be considered as different taxa with distinct host ranges.

Temporal isolation explains host‐related genetic differentiation in a group of widespread mycoparasitic fungi

Understanding the mechanisms responsible for divergence and specialization of pathogens on different hosts is of fundamental importance, especially in the context of the emergence of new diseases via host shifts. Temporal isolation has been reported in a few plants and parasites, but is probably one of the least studied speciation processes. We studied whether temporal isolation could be responsible for the maintenance of genetic differentiation among sympatric populations of Ampelomyces, widespread intracellular mycoparasites of powdery mildew fungi, themselves plant pathogens. The timing of transmission of Ampelomyces depends on the life cycles of the powdery mildew species they parasitize. Internal transcribed spacer sequences and microsatellite markers showed that Ampelomyces populations found in apple powdery mildew (Podosphaera leucotricha) were genetically highly differentiated from other Ampelomyces populations sampled from several other powdery mildew species across Europe, infecting plant hosts other than apple. While P. leucotricha starts its life cycle early in spring, and the main apple powdery mildew epidemics occur before summer, the fungal hosts of the other Ampelomyces cause epidemics mainly in summer and autumn. When two powdery mildew species were experimentally exposed to Ampelomyces strains naturally occurring in P. leucotricha in spring, and to strains naturally present in other mycohost species in autumn, cross‐infections always occurred. Thus, the host‐related genetic differentiation in Ampelomyces cannot be explained by narrow physiological specialization, because Ampelomyces were able to infect powdery mildew species they were unlikely to have encountered in nature, but instead appears to result from temporal isolation.

Variation in the nrDNA ITS sequences of some powdery mildew species: do routine molecular identification procedures hide valuable information?

During the past years, nrDNA ITS sequences have supported the identification of many powdery mildew fungi because comprehensive analyses showed that differences in these sequences have always correlated with the delimitation of different species and formae speciales of the Erysiphales. Published data, obtained using direct sequencing of the PCR products, suggested that even one to five nucleotide differences in the ITS sequences delimit different, albeit closely related, species, and/or indicate differences in host range patterns. Here we show that such differences in the ITS sequences can be detected even in a single sample of a powdery mildew fungus. We sequenced the ITS region in 17 samples, representing six powdery mildew species, both directly and after cloning the PCR products. Among these, samples of O. longipes exhibited two or three, samples of O. neolycopersici three or four, those of an Oidium sp. from Chelidonium majus up to seven, and a sample of another Oidium sp. from Passiflora caerulea two different ITS types determined after cloning. No ITS nucleotide polymorphisms were found in samples of O. lycopersici and Erysiphe aquilegiae. This suggests that some powdery mildew taxa are more variable at the ITS level than others. Thus, although the ITS sequences determined by direct sequencing represent robust data useful in delimitation and phylogenetic analysis of distinct species of the Erysiphales, these need to be used with precaution, and preferably determined after cloning, especially when dealing with closely related taxa at species and sub-species levels. With this method a hitherto undetected genetic diversity of powdery mildews can be revealed.

Microcyclic conidiogenesis in powdery mildews and its association with intracellular parasitism by Ampelomyces

Microcyclic conidiogenesis (MC), a process defined as the production of conidia on a spore without any, or only a minimal, involvement of hyphal growth, has recently been reported in a little known powdery mildew species, Oidium longipes. To investigate whether this was an isolated case or it is a more general phenomenon in powdery mildew fungi, germinating conidia of eight species of the Erysiphales were examined using light microscopy. The following species were included in this work: Erysiphe necator on grapevine, Blumeria graminis f. sp. hordei on barley, Podosphaera xanthii on cucumber, Erysiphe sp. on Ligustrum vulgare, O. longipes on Petunia x grandiflora, O. neolycopersici on tomato, Golovinomyces cichoracearum on Rudbeckia laciniata and Sawadaea sp. on Acer negundo. In all these species, up to 4% of the germinated conidia exhibited MC. Moreover, when colonies of E. necator and O. neolycopersici, on detached grapevine and tomato leaves, respectively, were treated with a conidial suspension of Ampelomyces, the intracellular pycnidia of these mycoparasites appeared in microcyclic conidiophores. This represents a yet undescribed method of accelerating asexual reproduction in this mycoparasite. In the life cycle of powdery mildews, the importance of MC is still not clear but it should be taken into consideration when conidial germination is studied on the host surface for purposes such as epidemiology or species identification.

New Insights into the Life Cycle of the Wheat Powdery Mildew: Direct Observation of Ascosporic Infection in Blumeria graminis f. sp. tritici

Although Blumeria graminis is an intensively studied pathogen, an important part of its life cycle (namely, the way ascospores initiate primary infections on cereal leaves) has not yet been explored in detail. This study reports, for the first time, the direct observation of this process in B. graminis f. sp. tritici using light and confocal laser-scanning microscopy. All the germinated ascospores produced a single germ tube type both in vitro and on host plant surfaces; therefore, the ascosporic and conidial germination patterns are markedly different in this fungus, in contrast to other powdery mildews. Germinated ascospores penetrated the epidermal cells of wheat leaves and produced haustoria as known in the case of conidial infections. This work confirmed earlier studies reporting that B. graminis chasmothecia collected from the field do not contain mature ascospores, only asci filled with protoplasm; ascospore development is induced by moist conditions and is a fast process compared with other powdery mildews. Although ascosporic infections are frequent in B. graminis f. sp. tritici in the field, as shown by this study and other works as well, a recent analysis of the genomes of four isolates revealed the signs of clonal or near-clonal reproduction. Therefore, chasmothecia and ascospores are probably more important as oversummering structures than genetic recombination factors in the life cycle of this pathogen.

Genetic diversity and host range of powdery mildews on Papaveraceae

Because of the strong morphological similarity of the powdery mildew fungi that infect papaveraceous hosts, a total of 39 samples were studied to reveal the phylogeny and host range of these fungi. ITS and 28S sequence analyses revealed that the isolates identified earlier as Erysiphe cruciferarum on papaveraceous hosts represent distinct lineages and differ from that of E. cruciferarum sensu stricto on brassicaceous hosts. The taxonomic status of the anamorph infecting Eschscholzia californica was revised, and therefore, a new species name, Erysiphe eschscholziae, is proposed. The taxonomic position of the Pseudoidium anamorphs infecting Glaucium flavum, Meconopsis cambrica, Papaver dubium, and Stylophorum diphyllum remain unclear. This study revealed that Erysiphe macleayae exhibits a specific host range different from that of E. cruciferarum, the common pathogen of papaveraceous hosts. Although E. macleayae occurred naturally on Macleaya cordata, Macleaya microcarpa, M. cambrica, and Chelidonium majus only, our inoculation tests revealed that the fungus was capable of infecting Argemone grandiflora, Glaucium corniculatum, Papaver rhoeas, and Papaver somniferum, indicating that these plant species may also be taken into account as potential hosts. Erysiphe cruciferarum originating from P. somniferum was not able to infect A. grandiflora, C. majus, E. californica, M. cordata, and P. rhoeas. The emergence of E. macleayae on M. microcarpa is reported here for the first time from the Czech Republic and Slovakia. The appearance of chasmothecia of E. macleayae on C. majus in Slovakia was reported, as well. Erysiphe cruciferarum was identified on G. corniculatum and reported here for the first time from Slovakia.

First Report of Powdery Mildew (Oidium sp.) on Pincushion Flower (Scabiosa columbaria) in New York

Scabiosa columbaria (Dipsacaceae) is a popular perennial ornamental in the United States. It is native to Europe and was introduced to North America by nursery trade only recently. In the spring of 2006, symptoms of powdery mildew infection were observed on overwintered plants of S. columbaria cv. Butterfly Blue in a nursery in Cutchogue, NY. White powdery mildew mycelia with abundant sporulation were observed on upper and lower leaf surfaces. The portions of leaves with powdery mildew colonies often showed purplish discoloration. Conidia were cylindric to doliiform, measured 20 to 33 × 10 to 15 μm, and were produced singly on 60 to 130 μm long conidiophores consisting of a foot-cell measuring 20 to 50 × 6 to 10 μm, followed by one to three, 12 to 40 μm long cells. Hyphal appressoria were lobed or multilobed. The teleomorph stage was not found. On the basis of these characteristics, the pathogen was identified as an Oidium sp. belonging to the subgenus Pseudoidium. Recently, an anamorphic powdery mildew fungus with similar morphological characteristics, identified as Erysiphe knautiae, was reported on S. columbaria cv. Butterfly Blue in Washington (2). E. knautiae is a common powdery mildew species of dipsacaceous plants such as Scabiosa spp. and Knautia spp. in Europe and Asia (1). To determine whether the fungus reported here was E. knautiae, DNA was extracted from its mycelium, and the internal transcribed spacer (ITS) region of the ribosomal DNA was amplified and sequenced as described earlier (4). No ITS sequences are available in public DNA databases for E. knautiae, thus, we determined this sequence in a specimen of E. knautiae collected from Knautia arvensis in The Netherlands. Herbarium specimens of the Oidium sp. infecting S. columbaria in New York and E. knautiae from the Netherlands were deposited at the U.S. National Fungus Collections under accession numbers BPI 878259 and BPI 878258, respectively. The ITS sequence from Oidium sp. infecting S. columbaria in New York (GenBank Accession No. EU377474) differed in two nucleotides from that of E. knautiae infecting K. arvensis in the Netherlands (GenBank Accession No. EU377475). These two ITS sequences were also more than 99% similar to those of some newly emerged anamorphic powdery mildew fungi: Oidium neolycopersici and other Oidium spp. infecting Chelidonium majus, Passiflora caerulea, and some crassulaceous plants (3,4). Thus, it is unclear whether the fungus reported here was E. knautiae known from Eurasia or an Oidium sp. that has acquired pathogenicity to S. columbaria. To our knowledge, this is the first report of powdery mildew on S. columbaria in New York. References: (1) U. Braun. Beih. Nova Hedwigia 89:1, 1987. (2) D. A. Glawe and G. G. Grove. Online publication. doi:10.1094/PHP-2005-1024-01-BR. Plant Health Progress, 2005. (3) B. Henricot. Plant Pathol. 57:779, 2008. (4) T. Jankovics et al. Phytopathology 98:529, 2008.