Molecular phylogenetic and morphological analyses of Oidium heveae, a powdery mildew of rubber tree

Powdery mildew of rubber tree caused by Oidium heveae is an important disease of rubber plantations worldwide. Identification and classification of this fungus is still uncertain because there is no authoritative report of its morphology and no record of its teleomorphic stage. In this study, we compared five specimens of the rubber powdery mildew fungus collected in Malaysia, Thailand, and Brazil based on morphological and molecular characteristics. Morphological results showed that the fungus on rubber tree belongs to Oidium subgen. Pseudoidium. Nucleotide sequence analysis of the ribosomal DNA internal transcribed spacer (ITS) region and the large subunit rRNA gene (28S rDNA) were conducted to determine the relationships of the rubber powdery mildew fungus and to link this anamorphic fungus with its allied teleomorph. The results showed that the rDNA sequences of the two specimens from Malaysia were identical to a specimen from Thailand, whereas they differed by three bases from the two Brazilian isolates: one nucleotide position in the ITS2 and two positions in the 28S sequences. The ITS sequences of the two Brazilian isolates were identical to sequences of Erysiphe sp. on Quercus phillyraeoides collected in Japan, although the 28S sequences differed at one base from sequences of this fungus. Phylogenetic trees of both rDNA regions constructed by the distance and parsimony methods showed that the rubber powdery mildew fungus grouped with Erysiphe sp. on Q. phillyraeoides with 100% bootstrap support. Comparisons of the anamorph of two isolates of Erysiphe sp. from Q. phillyraeoides with the rubber mildew did not reveal any obvious differences between the two powdery mildew taxa, which suggests that O. heveae may be an anamorph of Erysiphe sp. on Q. phillyraeoides. Cross-inoculation tests are required to substantiate this conclusion.     

Identity of a powdery mildew fungus occurring on Paeonia and its relationship with Erysiphe hypophylla on oak

A powdery mildew fungus found on Paeonia lutea at the Botanical Garden of Geneva (Switzerland) was identified as Erysiphe hypophylla based on morphological observations. The occurrence of E. hypophylla on Paeonia seemed curious, because host plants of this species have been restricted to a few Quercus species of the family Fagaceae. In this study, we determined the rDNA sequences of the powdery mildew specimens on Paeonia and E. hypophylla on Quercus to confirm the identity of the Paeonia fungus. The three sequences from the specimens on P. lutea were identical to one another in both ITS and 28S rDNA regions and also to the sequences of E. hypophylla on Q. robur, which supports the identification that the fungus on P. lutea is E. hypophylla. However, these sequences were also identical to the sequences of E. alphitoides on Quercus spp. and Oidium mangiferae on mango. This result suggests a possibility that E. hypophylla is conspecific to E. alphitoides. Further study is required to clarify whether E. hypophylla is a synonym of E. alphitoides or a distinct species.     

Phylogeny and taxonomy of powdery mildew fungi of Erysiphe sect. Uncinula on Carpinus species

A phylogenetic analysis of the Erysiphe with uncinuloid ascoma appendages (Erysiphe section Uncinula, Erysiphales, Ascomycota) on Carpinus spp. was done using sequences of the rDNA ITS regions and the D1/D2 domains of the 28S rDNA. These results, combined with morphological data, revealed a complex consisting of several distinct taxa. These included the already described Erysiphe carpinicola on C. japonica distinguishable from the Erysiphe sp. on C. betulus and C. tschonoskii as well as the one on C. laxiflora. Thus, it was shown that Oidium carpini, described from Europe on Carpinus betulus, the powdery mildew with uncinula-like ascomata, recently found in Europe on this host, as well as an Erysiphe on C. tschonoskii in Japan, described previously as E. carpinicola, all belong to a single new species, named E. arcuata in this paper. As the powdery mildew on C. laxiflora was also distinct from other known species, it is named E. carpini-laxiflorae in this paper. The already described E. pseudocarpinicola and Erysiphe sp. on Carpinus cordata are two additional taxa, which are morphologically and genetically distinguished from the other species of Erysiphe sect. Uncinula on Carpinus spp.     

Molecular data do not support a southern hemisphere base of Nothofagus powdery mildews

Three powdery mildew species present on Nothofagus (viz. Erysiphe magellanica, E. nothofagi and E. patagoniaca) are endemic to South America and have unique ascomatal appendages that are not found in powdery mildews of the northern hemisphere. We determined the nucleotide sequences of the rDNA internal transcribed spacer regions and D1/D2 domains of the 28S rDNA of these three powdery mildew species to reveal their phylogenetic relationships with powdery mildews of the northern hemisphere. Although the molecular phylogenetic analyses indicated that the three Nothofagus powdery mildews are closely related to each other they did not group into one clade in either the ITS or 28S trees. Kishino-Hasegawa, Shimodaira-Hasegawa and Templeton tests could not significantly reject the constrained trees that were constructed based on the assumption that the Nothofagus powdery mildews would form a single clade. Based on this result and the evidence that all Nothofagus powdery mildews are endemic to South America and have similar morphological characteristics, it is likely that these three species diverged from a single ancestor present on Nothofagus. Calibration of evolutionary events with molecular clocks suggested that the Nothofagus powdery mildews split from the northern hemisphere relatives 22-16 million y ago (Ma) in the middle Miocene, and divergence among the Nothofagus powdery mildews occurred 17-13 Ma. These results do not support a southern hemisphere base of the Nothofagus powdery mildews.     

Interspecific and intraspecific diversity in oak powdery mildews in Europe: coevolution history and adaptation to their hosts

Quercus has been reported as the genus with the largest number of attacking powdery mildews. In Europe, oak powdery mildew was rarely reported before 1907, when severe outbreaks were observed. These epidemics were attributed to the newly described species Erysiphe alphitoides, presumed to be of exotic origin. After the burst of interest following the emergence of the disease, research on this topic remained very limited. Interest in research was recently reactivated in response to the availability of molecular tools. This review summarizes current knowledge on the diversity of oak powdery mildews in Europe and their possible evolutionary relationships with European oaks. The most striking results are the evidence of cryptic diversity (detection in France of a lineage closely related to Erysiphe quercicola, previously thought to only have an Asian distribution), large host range (similarity of E. alphitoides and E. quercicola with powdery mildews of tropical plants) but also local adaptation to Quercus robur. These recent findings highlight the complexity of the history of oak powdery mildew in Europe and point to the question of host specialization and host jumps in the evolution of powdery mildew fungi.     

A new subgenus, Microidium, of Oidium (Erysiphaceae) on Phyllanthus spp.

Powdery mildew fungi found on leaves, shoots, and stems of Phyllanthus acidus, P. amarus, and P. reticulatus proved to be a fungus having morphology unique in the Erysiphaceae. Light micrographs of a new germination pattern are added to discuss differences to other four germination patterns of the powdery mildews. The rDNA sequences (28S and 18S regions) of the fungi found on Phyllanthus spp. form a distinct monophyletic clade strongly supported by bootstrap (100%) in 18S + 28S trees, which indicates that the fungus is an isolated fungal group among the Erysiphaceae in tribal level. Because we cannot find the teleomorphic state of this fungus, a new subgenus Microidium of anamorphic genus Oidium is proposed to accommodate this organism.     

Phylogenetic relationships ofMicrosphaera andErysiphe sectionErysiphe (powdery mildews) inferred from the rDNA ITS sequences

The genusMicrosphaera has been considered to be derived from sectionErysiphe of the genusErysiphe by a single event. Cleistothecial appendages are the most distinct difference between the two genera and have an important role for overwintering. To understand the phylogenetic relationship betweenErysiphe sectionErysiphe andMicrosphaera more precisely, phylogenetic trees were constructed using the nucleotide sequences of the rDNA ITS region from 11Erysiphe (sectionErysiphe) and 16Microsphaera taxa. The phylogenetic trees indicated the close relationship between the two genera. However, the generaErysiphe (sectionErysiphe) andMicrosphaera did not group into separate monophyletic lineages; instead, they formed several small clusters that were mixed together. This result suggests that the differentiations of the genera occurred two or more times independently. This also supports the idea that appendage morphology does not always accurately reflect the phylogeny of the powdery mildews because the morphology of appendages may evolve convergently under the selection pressure of their particular biotopes (host plants). Contribution No. 144 from the Laboratory of Plant Pathology, Mie University.     

Nucleotide sequence diversity of rDNA internal transcribed spacers extracted from conidia and cleistothecia of several powdery mildew fungi

An improved protocol, including DNA extraction with Chelex, two amplifications with a nested primer set, and DNA purification by electrophoresis, made it possible to analyze nuclear rDNA sequences of powdery mildew fungi using at most several hundred conidia or 20 cleistothecia. Nucleotide sequence diversity of the nuclear rDNA region containing the two internal transcribed spacers (ITS1 and ITS2) and 5.8S rRNA gene derived from conidia and cleistothecia was investigated for four kinds of powdery mildew fungi including two isolates of the same species. The results showed that the nucleotide sequences of the nuclear rDNA region were highly conserved between the teleomorph and the anamorph. Thus, the nucleotide sequence data obtained from either developmental stage can be used for phylogenetic studies of powdery mildew fungi. The nucleotide sequences of the 5.8S rRNA genes of the four species were highly conserved, but those of their ITS regions were variable. This suggests that the nuclear rDNA region is not suitable for phylogenetic studies of distantly related powdery mildew fungi, because too much sequence diversity exists, within the ITS, and too little phylogenetic information is contained within the 5.8S rRNA gene. However, the ITS region will be useful for phylogenetic comparison of closely related species or intraspecies. Contribution No. 132 from the Laboratory of Plant Pathology, Mie University.     

PCR primers useful for nucleotide sequencing of rDNA of the powdery mildew fungi

Four PCR primers that are useful to determine the nucleotide sequences of the rDNA of the powdery mildew fungi were newly designed. These primers provide both enough stability to work on a wide range of powdery mildews and enough specificity to eliminate contaminating DNA by PCR. DNA sequences of the rDNA ITS region were successfully obtained from specimens that were contaminated by other fungi. In addition, sequence results of the 18S and 28S rDNA were dramatically improved by using these primers in most of the specimens examined.     

Phylogenetic analysis and predicted secondary structures of the rDNA internal transcribed spacers of the powdery mildew fungi (Erysiphaceae)

The nucleotide sequences of the internal transcribed spacer (ITS) regions of the ribosomal DNA including the 5.8S rRNA gene and the 5′ end of the 28S rRNA gene have been determined for 19 species in 10 genera of the powdery mildew fungi in order to analyze their phylogenetic relationship. These fungi were divided into two large groups based on the nucleotide length of the ITS regions, and this grouping was in line with that based on the morphological characters of the anamorphic stage rather than the teleomorphic stage. Although the variable ITS sequences were often ambiguously aligned, conserved sites were also found. Thus, a neighbor-joining tree was constructed using the nucleotide sequence data of the conserved sites of the ITS regions, the 5.8S rRNA gene, and the 5′ end of the 28S rRNA gene. The phylogenetic tree displayed the presence of four groups in the powdery mildews, which were distinguished by their morphology and/or host ranges. In the ITS2 region, the presence of a common secondary structure having four hairpin domains was suggested, in spite of the highly variable nucleotide sequences of this region. The predicted secondary structure was supported by the compensatory mutations as well as compensatory conserved sequences and high G+C content in the predicted stem regions. Contribution No. 142 from the Laboratory of Plant Pathology, Mie University.     

Genetic variability and mycohost association of Ampelomyces quisqualis isolates inferred from phylogenetic analyses of ITS rDNA and actin gene sequences

Ampelomyces quisqualis complex is well known as the most common and widespread hyperparasite of the family Erysiphaceae, the cause of powdery mildew diseases. As commercial biopesticide products it is widely used to control the disease in field and plastic houses. Although genetic diversity within Ampelomyces isolates has been previously recognized, a single name A. quisqualis is still applied to all pycnidial intracellular hyperparasites of powdery mildew fungi. In this study, the phylogenetic relationships among Ampelomyces isolates originating from various powdery mildew fungi in Korea were inferred from Bayesian and maximum parsimony analyses of the sequences of ITS rDNA region and actin gene. In the phylogenetic trees, the Ampelomyces isolates could be divided into four distinct groups with high sequence divergences in both regions. The largest group, Clade 1, mostly accommodated Ampelomyces isolates originating from the mycohost Podosphaera spp. (sect. Sphaerotheca). Clade 2 comprised isolates from several genera of powdery mildews, Golovinomyces, Erysiphe (sect. Erysiphe), Arthrocladiella, and Phyllactinia, and was further divided into two subclades. An isolate obtained from Podosphaera (sect. Sphaerotheca) pannosa was clustered into Clade 3, with those from powdery mildews infecting rosaceous hosts. The mycohosts of Ampelomyces isolates in Clade 4 mostly consisted of species of Erysiphe (sect. Erysiphe, sect. Microsphaera, and sect. Uncinula). The present phylogenetic study demonstrates that Ampelomyces hyperparasite is indeed an assemblage of several distinct lineages rather than a sole species. Although the correlation between Ampelomyces isolates and their mycohosts is not obviously clear, the isolates show not only some degree of host specialization but also adaptation to their mycohosts during the evolution of the hyperparasite.     

Characterization of six polymorphic microsatellite loci from Ampelomyces quisqualis,intracellular mycoparasite and biocontrol agent of powdery mildew

Ampelomyces spp. are common intracellular mycoparasites of powdery mildews worldwide and a strain has been commercialized as a biocontrol agent against these plant pathogens. In light of recent genetic analyses revealing high internal transcribed spacer sequence variability among Ampelomyces strains on different host plant mildews, yet no sequence variability within the Malus strain, polymorphic microsatellites were required to permit biocontrol and ecological studies of the complex apple/apple mildew/Ampellomyces tritrophic interaction. For this purpose, described here are the primers to amplify six polymorphic microsatellite loci from Ampelomyces quisqualis isolated from mycelia of the apple powdery mildew fungus, Podosphaera leucotricha.     

The effectivity of Tilletiopsis albescens in biocontrol of powdery mildew

Tilletiopsis albescens grows well on powdery mildew fungi inoculated on barley or cucumber leaves and causes collapse of the colonies. Application of ballistospores or cut mycelium was equally effective for biocontrol, and the effectiveness tended to increase exponentially with the concentration of germinating units (conidia and cut mycelium) applied. Seventy percent relative humidity or more is required for effective biocontrol. Two applications of T. albescens in the period from 3 days before to 3 days after inoculation with powdery mildew were more effective than one. Applications before inoculation or 7 days after inoculation with powdery mildew had little effect. T. albescens followed the powdery mildew as it was disseminated to uninoculated leaves, but this did not result in an effective biocontrol. The potential for using T. albescens for biocontrol of powdery mildews is discussed.     

Four powdery mildew species with catenate conidia infect Galium: molecular and morphological evidence

The Erysiphaceae are a group of obligately biotrophic fungi that cause powdery mildew disease of angiosperms. Due to their inability to be cultured on artificial media, the taxonomy of the Erysiphaceae has generally been based on the morphological characteristics of fresh and herbarium specimens. Thus, several morphological species with wide host ranges have long been maintained in this family, even though they clearly consist of several biological species. Erysiphe galii has been known as a powdery mildew of Galium spp. Recently, the former E. galii var. galii has been reassessed as Neoerysiphe galii and E. galii var. riedliana as Golovinomyces riedlianus, along with a taxonomic revision of the generic concept of the Erysiphaceae. The present study was conducted to evaluate the validity of the taxonomic revision of the two varieties of E. galii. During the course of this study, we found that the Galium powdery mildews consist of at least four different species, viz. Neoerysiphe galii, Golovinomyces orontii, G. riedlianus, and an unknown species collected in Argentina. The latter species is described as a new species, Golovinomyces calceolariae. The three species belonging to Golovinomyces are morphologically very similar to each other, i.e. the discrimination between them is rather difficult. The morphological differences of the three Golovinomyces species of Galium are discussed.     

De novo indol-3-ylmethyl glucosinolate biosynthesis,and not long-distance transport,contributes to defence of Arabidopsis against powdery mildew

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolate is constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolate is activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis demonstrate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.     

Multilocus phylogenetic analyses within Blumeria graminis, a powdery mildew fungus of cereals

Blumeria graminis, a powdery mildew fungus, is an important plant pathogen that causes serious damage to a variety of cereal crops. In spite of the importance of the pathogen, information on phylogenetic structure within B. graminis is scarce. In this study we conducted phylogenetic analyses of B. graminis based on the DNA sequences of four different DNA regions (ITS, 28S rDNA, chitin synthase 1, and beta-tubulin). The analyses revealed that the protein-coding regions have higher amounts of phylogenetic signals than rDNA regions and are useful for phylogenetic analyses of B. graminis. The present phylogenetic analyses revealed nine distinct groups in the B. graminis isolates used in this study, a result which was commonly supported by all trees constructed from the four DNA regions. Isolates from a single host genus belonged to a single group except for isolates from Lolium and Bromus, in which the isolates were split into two and three groups, respectively. Isolates from Agropyron, Secale and Triticum formed a distinct clade (Triticum clade) with identical or similar DNA sequences. The Hordeum clade was a sister of the Triticum clade, and Poa and Avena clades were distantly related to the Triticum and Hordeum clades. This phylogenetic relationship of B. graminis is well concordant with the level of reproductive isolation between formae speciales and also with phylogeny inferred from a cytological study. Shimodaira-Hasegawa and Templeton tests using sequences of four different DNA regions significantly rejected the tree topology of plants. Therefore, possibility of co-speciation between B. graminis and its host plants was obscure in this study.     

Community of arbuscular mycorrhizal fungi in drought-resistant plants, <Emphasis Type="Italic">Moringa</Emphasis> spp., in semiarid regions in Madagascar and Uganda

The community structure of arbuscular mycorrhizal (AM) fungi in the roots of drought-resistant trees, Moringa spp., was examined in semiarid regions in Madagascar and Uganda. Root samples were collected from 8 individuals of M. hildebrandtii and 2 individuals of M. drouhardii in Madagascar and from 21 individuals of M. oleifera in Uganda. Total DNA was extracted from the root samples, and partial nSSU rDNA of AM fungi was amplified using a universal eukaryotic primer NS31 and an AM fungalspecific primer AM1. The PCR products were cloned and divided by restriction fragment length polymorphism (RFLP) analysis with HinfI and RsaI. Some representatives in each RFLP types were sequenced, and a neighbor-joining phylogenetic analysis was conducted for the obtained sequences with analogous sequences of AM fungi. The RFLP and phylogenetic analyses showed that AM fungi closely related to Glomus intraradices or G. sinuosum were detected in many samples. The AM fungal groups frequently detected in the Moringa spp. might be widely distributed species in semiarid environments.     

Phylogeny and evolution of the powdery mildew fungi (Erysiphales,Ascomycota) inferred from nuclear ribosomal DNA sequences

Powdery mildew fungi (Erysiphales, Ascomycota) are obligate biotrophs that infect a wide range of angiosperms. Phylogenetic analyses based on the nucleotide sequences of the nuclear ribosomal DNA revealed that the powdery mildew fungi are divided into five major lineages. The respective lineage was well defined by the morphology of conidial stage, but not of ascomata. In this fungal group, tree-parasitic taxa are generally ancestral, and multiple events of host expansion from trees to herbs have occurred within the respective lineage. Accompanying the host expansion to herbs, simplification of appendage morphology has occurred multiple times. The simple, mycelioid appendages are thus a result of convergence. The host expansion route was investigated in detail in the tribe Cystotheceae. Two sections of the genus Sphaerotheca were derived from the genus Podosphaera separately. The section Magnicellulatae was derived from a Podosphaera species parasitic to Prunus (Rosaceae), acquired parasitism to the Scrophulariaceae, and then expanded host range into the Asteraceae. After genetic radiation on the Asteraceae, they further expanded their host ranges into other plant families. According to the molecular clock (1.26%/100 million years, myr) reported by Berbee and Taylor, splitting of the Erysiphales and the Myxotrichaceae and first divergence within the Erysiphales was calculated to have occurred 100 and 76myr ago, respectively.     

Erysiphe kenjiana (Erysiphales), a new invasive fungus in Europe

An Asian powdery mildew fungus Erysiphe (Uncinula) kenjiana (Erysiphales, Ascomycota) has been found in Ukraine. This is the first record of this fungus in Europe. In 2007, E. kenjiana was collected on four Ulmus species in Kiev. All locations adjoined railways or an airport. Development of E. kenjiana was epiphytotic. This species was not found on elms surveyed at towns situated north-east, east or south of Kiev. The fungus may have been brought directly to Ukraine by rail or air transport. In 2008, the fungus was also collected in Chernihiv situated north-east of Kiev. It is likely that E. kenjiana will spread over all Ukraine and into countries of central and western Europe in 2009 or later. Molecular phylogenetic analyses using D1/D2 domains of the 28S rDNA and ITS sequences revealed that the Ulmaceae-Cannabaceae-parasitic powdery mildews, including E. kenjiana, form a clade with strong supports, suggesting that these species diverged from a single ancestor and expanded their host ranges within the Ulmaceae and allied Cannabaceae. This hypothesis is supported by these species sharing the unique morphology of enlarged apices on their chasmothecial appendages. These fungi formed part of a larger grouping with species on Fagaceae, Nothofagaceae, Rosaceae, and Sapindaceae with strong statistical supports. These results suggest that Uncinula-like powdery mildew fungi on these plant families exhibit close evolutionary relationships with their hosts.