<Emphasis Type="Italic">BrRxLR11</Emphasis> – a new phylogenetic marker with high resolution in the downy mildew genus <Emphasis Type="Italic">Bremia</Emphasis> and related genera

The genus Bremia (Peronosporaceae; Oomycete) is a widespread pathogen of Eurasian Asteraceae (Compositae). In addition to several species infecting weeds, it includes the economically important species, B. lactucae, which causes downy mildew of lettuce (Lactuca sativa). A few loci have already been successfully sequenced for this genus, but the resolution of the resultant phylogenies is insufficient to resolve the evolutionary history of Bremia. Therefore, there is a need to develop additional loci that can provide high resolution in phylogenetic inference. In this study, we report a new locus, BrRxLR11, derived from genomic data, which shows high resolving power within the genus Bremia, similar to phylogenies based on three different loci. This gene encodes a protein of 204 aa with unknown function, a below-threshold secretion signal, and an RxLR-EER motif in the c-terminal half. As only one site seems to be under positive selection, it has potential for future application in phylogenetic investigations in the economically important genus Bremia and related genera.     

A revision of Plasmopara penniseti, with implications for the host range of the downy mildews with pyriform haustoria

Plasmopara penniseti is the sole member of the genus Plasmopara parasitic to Poaceae, after the genus Viennotia had been described to accommodate Plasmopara oplismeni. Morphological, ultrastructural, and molecular phylogenetic data indicate that Plasmopara penniseti is not closely related to the generic type, and it is, therefore, transferred to the newly described genus Poakatesthia. The view that the genera of downy mildews with pyriform to vesicular haustoria (Basidiophora, Benua, Bremia, Paraperonospora, Plasmopara, Plasmoverna, and Protobremia) include species parasitic to Poaceae has to be discarded. All of these genera are apparently restricted to dicotyledonous hosts.     

DNA fingerprinting detects genetic variability in the pearl millet downy mildew pathogen (Sclerospora graminicola)

Genetic variability in six host genotype-specific pathotypes of pearl millet downy mildew pathogen S. graminicola was studied at the molecular level using mini- and micro-satellites. Our results indicated that microsatellites (GAA)₆, (GACA)₄, and especially (GATA)₄ were quite informative and showed high levels of polymorphism among the pathotypes. The six pathotypes could be classified into five groups based on the cluster analysis of their genetic similarities, thereby confirming the existence of distinct host genotype-specific virulence in S. graminicola pathotypes. We demonstrate, for the first time, the use of DNA fingerprinting to detect genetic variation in downy mildew fungus of pearl millet.     

Seed treatment with aqueous extract of Viscum album induces resistance to pearl millet downy mildew pathogen

Abstract

Downy mildew (Sclerospora graminicola [Sacc.] Schroet.) is a serious agricultural problem for pearl millet (Pennisetum glaucum [L.] R. Br.) grain production under field conditions. Six medicinally important plant species Azadirachta indica, Argemone mexicana, Commiphora caudata, Mentha piperita, Emblica officinalis and Viscum album were evaluated for their efficacy against pearl millet downy mildew. Seeds of pearl millet were treated with different concentrations of aqueous extract of the plants to examine their efficacy in controlling downy mildew. Among the plant extracts tested, V. album treatment was found to be more effective in enhancing seed quality parameters and also in inducing resistance against downy mildew disease. Germination and seedling vigor was improved in seeds treated with V. album extracts over control. Seeds treated with 10% concentration of V. album showed maximum protection against downy mildew disease under greenhouse and field conditions. The downy mildew disease protection varied from 44–70% with different concentrations. Leaf extract of V. album did not inhibit sporulation and zoospore release from sporangia of Sclerospora graminicola, indicating that the disease-controlling effect was attributed to induced resistance. Seed treatment with V. album extract increased pearl millet grain yield considerably. In V. album, treated pearl millet seedlings increased activities of peroxidase, and phenylalanine ammonia-lyase enzyme was detected. FTIR analysis of V. album extracts showed the presence of amides and other aromatic compounds which are antimicrobial compounds involved in plant defense.     

Phylogenetic Analyses of Meloidogyne Small Subunit rDNA

Phylogenies were inferred from nearly complete small subunit (SSU) 18S rDNA sequences of 12 species of Meloidogyne and 4 outgroup taxa (Globodera pallida, Nacobbus abberans, Subanguina radicicola, and Zygotylenchus guevarai). Alignments were generated manually from a secondary structure model, and computationally using ClustalX and Treealign. Trees were constructed using distance, parsimony, and likelihood algorithms in PAUP* 4.0b4a. Obtained tree topologies were stable across algorithms and alignments, supporting 3 clades: clade I = [M. incognita (M. javanica, M. arenaria)]; clade II = M. duytsi and M. maritima in an unresolved trichotomy with (M. hapla, M. microtyla); and clade III = (M. exigua (M. graminicola, M. chitwoodi)). Monophyly of [(clade I, clade II) clade III] was given maximal bootstrap support (mbs). M. artiellia was always a sister taxon to this joint clade, while M. ichinohei was consistently placed with mbs as a basal taxon within the genus. Affinities with the outgroup taxa remain unclear, although G. pallida and S. radicicola were never placed as closest relatives of Meloidogyne. Our results show that SSU sequence data are useful in addressing deeper phylogeny within Meloidogyne, and that both M. ichinohei and M. artiellia are credible outgroups for phylogenetic analysis of speciations among the major species.     

Species delimitation in downy mildews: the case of Hyaloperonospora in the light of nuclear ribosomal ITS and LSU sequences

Species definitions for plant pathogens have considerable practical impact for measures such as plant protection or biological control, and are also important for comparative studies involving model organisms. However, in many groups, the delimitation of species is a notoriously difficult taxonomic problem. This is particularly evident in the obligate biotrophic downy mildew genera (Peronosporaceae, Peronosporales, Oomycetes), which display a considerable diversity with respect to genetic distances and host plants, but are, for the most part, morphologically rather uniform. The recently established genus Hyaloperonospora is of particular biological interest because it shows an impressive radiation on virtually a single host family, Brassicaceae, and it contains the downy mildew parasite, Arabidopsis thaliana, of importance as a model organism. Based on the most comprehensive molecular sampling of specimens from a downy mildew genus to date, including various collections from different host species and geographic locations, we investigate the phylogenetic relationships of Hyaloperonospora by molecular analysis of the nuclear ribosomal ITS and LSU sequences. Phylogenetic trees were inferred with ML and MP from the combined dataset; partitioned Bremer support (PBrS) was used to assess potential conflict between data partitions. As in other downy mildew groups, the molecular data clearly corroborate earlier results that supported the use of narrow species delimitations and host ranges as taxonomic markers. With few exceptions, suggested species boundaries are supported without conflict between different data partitions. The results indicate that a combination of molecular and host features is a reliable means to discriminate downy mildew species for which morphological differences are unknown.     

<Emphasis Type="Italic">Basidiophora delawarensis</Emphasis>, a new downy mildew species infecting cultivated goldenrod (<Emphasis Type="Italic">Solidago sphacelata</Emphasis>) in the USA

Species in the genus Basidiophora (Oomycota, Peronosporales) are pathogens causing downy mildew disease on several closely related plant hosts in the family Asteraceae, including the genera Conyza, Erigeron, Solidago, and Symphyotrichum. Despite their widespread occurrence, little is known about the diversity, biology, and economic impact of downy mildew pathogens of ornamental or wild plants in the Asteraceae. In June 2017, Solidago sphacelata plants of the cultivar ‘Golden Fleece’ were found in the state of Delaware, USA, showing typical symptoms of downy mildew disease. Initial morphological observations indicated the specimen belonged to the genus Basidiophora. Phylogenetic analysis of a sequence matrix of the partial cox2 mitochondrial gene using Bayesian and maximum likelihood methods showed that the Basidiophora isolate from S. sphacelata represents an undescribed species that is here described as B. delawarensis. This new species constitutes the third accepted species in the genus Basidiophora and can be distinguished from other Basidiophora species by differences in size of sporangia and sporangiophores and nucleotide sequence divergence. This new species poses a potential threat to its host, warranting close monitoring and the implementation of disease management programs.     

Reclassification of an enigmatic downy mildew species on lovegrass (Eragrostis) to the new genus Eraphthora, with a key to the genera of the Peronosporaceae

Graminicolous downy mildews affect economical important crops like sorghum, maize and pearl millet, and also a variety of wild grasses, which pathogens have hitherto been much less studied. This also applies to an enigmatic downy mildew from Eragrostis cilianensis, which exhibits a chimeric appearance, with similarities to Basidiophora, Sclerophthora, and Sclerospora. In this study, the phylogenetic relationship of lovegrass downy mildew with respect to other genera of the Peronosporaceae was investigated and the characteristics of genera potentially associated with lovegrass downy mildew summarised from historic studies. A key based on the morphology of asexual stages is presented for all genera of downy mildews hitherto described. Based on morphological characteristics and sequence divergence, Sclerospora butleri is transferred to a new genus, Eraphthora. Lovegrass downy mildew seems to be widely distributed, as in addition to previous records from Malawi, India and Australia, we could now sample one specimen from Europe (Spilimbergo, Italy). Currently, it is unclear if Eraphthora butleri is able to infect the crop species Eragrostis tef, which might be a potential host of economic importance.     

Which Morphological Characteristics Are Most Influenced by the Host Matrix in Downy Mildews? A Case Study in Pseudoperonospora cubensis

Before the advent of molecular phylogenetics, species concepts in the downy mildews, an economically important group of obligate biotrophic oomycete pathogens, have mostly been based upon host range and morphology. While molecular phylogenetic studies have confirmed a narrow host range for many downy mildew species, others, like Pseudoperonospora cubensis affect even different genera. Although often morphological differences were found for new, phylogenetically distinct species, uncertainty prevails regarding their host ranges, especially regarding related plants that have been reported as downy mildew hosts, but were not included in the phylogenetic studies. In these cases, the basis for deciding if the divergence in some morphological characters can be deemed sufficient for designation as separate species is uncertain, as observed morphological divergence could be due to different host matrices colonised. The broad host range of P. cubensis (ca. 60 host species) renders this pathogen an ideal model organism for the investigation of morphological variations in relation to the host matrix and to evaluate which characteristics are best indicators for conspecificity or distinctiveness. On the basis of twelve morphological characterisitcs and a set of twelve cucurbits from five different Cucurbitaceae tribes, including the two species, Cyclanthera pedata and Thladiantha dubia, hitherto not reported as hosts of P. cubensis, a significant influence of the host matrix on pathogen morphology was found. Given the high intraspecific variation of some characteristics, also their plasticity has to be taken into account. The implications for morphological species determination and the confidence limits of morphological characteristics are discussed. For species delimitations in Pseudoperonospora it is shown that the ratio of the height of the first ramification to the sporangiophore length, ratio of the longer to the shorter ultimate branchlet, and especially the length and width of sporangia, as well as, with some reservations, their ratio, are the most suitable characteristics for species delimitation.     

Downy mildew incidence of pearl millet hybrids with different male-sterility inducing cytoplasms

The use of different sources of cytoplasmic male sterility (CMS) in hybrid seed production of pearl millet [Pennisetum glaucum (L.) R. Br.] is advocated to avoid possible disease epidemics occurring due to cytoplasmic uniformity. The effects of commercially unexploited, but potentially exploitable, sources of CMS, like A₂, A₃ and A₄, on downy mildew [Sclerospora graminicola (Sacc.) Schroet] incidence were studied by using the disease incidence of isonuclear hybrids with male-sterile and fertile cytoplasm. The mean downy mildew incidence of hybrids carrying different male-sterile cytoplasm was similar to that of hybrids retaining the fertile cytoplasm. The cytoplasm accounted for only 0.6% of the total variation and its effect was non-significant; pollinators could explain most of the variation in determining the disease incidence of hybrids. This suggested that these male-sterile cytoplasms are not linked to downy mildew susceptibility and thus can be exploited commercially to broaden the cytoplasmic base of the male-sterile lines and, ultimately, of hybrids.     

Inheritance of downy mildew resistance, β-1,3-glucanases and peroxidases in pearl millet [Pennisetum glaucum (L.) R. Br.] crosses

The inheritance of resistance to downy mildew disease and the defense-related enzymes β-1,3-glucanase and peroxidase was studied in crosses of pearl millet using a generation-mean analysis. The study material comprised six generations (susceptible and resistant parents, F₁, F₂, BC₁ and BC₂) in three crosses. Seedlings from these generations were inoculated with the downy mildew pathogen Sclerospora graminicola and disease incidence was recorded. Analysis of constitutive levels of β-1,3-glucanase and peroxidase in the seedlings of different generations indicated that the resistant populations showed higher enzyme activities, while lower activities of the enzymes were recorded in the susceptible populations. In the generation-mean analysis, the significance of scaling tests revealed the existence of non-allelic interactions in the inheritance of resistance to downy mildew as well as with the enzymes. Among the gene effects, both additive and dominant effects were significant. All the non-allelic interaction effects were significant in the crosses. Studies on the isozyme patterns of the enzymes substantiated the results of the disease-incidence experiments in most of the generations. The results indicated that the inheritance of downy mildew disease resistance and the expression of β-1,3-glucanase and peroxidase in pearl millet is not only under the control of additive and dominant genes but are also governed by complex non-allelic interactions. Received: 30 April 2000 / Accepted: 17 October 2000     

Development of transgenic pearl millet (Pennisetum glaucum (L.) R. Br.) plants resistant to downy mildew

Transgenic pearl millet lines expressing pin gene—exhibiting high resistance to downy mildew pathogen, Sclerospora graminicola—were produced using particle-inflow-gun (PIG) method. Shoot-tip-derived embryogenic calli were co-bombarded with plasmids containing pin and bar genes driven by CaMV 35S promoter. Bombarded calli were cultured on MS medium with phosphinothricin as a selection agent. Primary transformants 1T₀, 2T₀, and 3T₀ showed the presence of both bar and pin coding sequences as evidenced by PCR and Southern blot analysis, respectively. T₁ progenies of three primary transformants, when evaluated for downy mildew resistance, segregated into resistant and susceptible phenotypes. T₁ plants resistant to downy mildew invariably exhibited tolerance to Basta suggesting co-segregation of pin and bar genes. Further, the downy mildew resistant 1T₁ plants were found positive for pin gene in Southern and Northern analyses thereby confirming stable integration, expression, and transmission of pin gene. 1T₂ progenies of 1T₀ conformed to dihybrid segregation of 15 resistant:1 susceptible plants.     

The phylogenetic relationships and generic limits of finches (Fringillidae)

Phylogenetic relationships among the true finches (Fringillidae) have been confounded by the recurrence of similar plumage patterns and use of similar feeding niches. Using a dense taxon sampling and a combination of nuclear and mitochondrial sequences we reconstructed a well resolved and strongly supported phylogenetic hypothesis for this family. We identified three well supported, subfamily level clades: the Holoarctic genus Fringilla (subfamly Fringillinae), the Neotropical Euphonia and Chlorophonia (subfamily Euphoniinae), and the more widespread subfamily Carduelinae for the remaining taxa. Although usually separated in a different family-group taxon (Drepanidinae), the Hawaiian honeycreepers are deeply nested within the Carduelinae and sister to a group of Asian Carpodacus. Other new relationships recovered by this analysis include the placement of the extinct Chaunoproctus ferreorostris as sister to some Asian Carpodacus, a clade combining greenfinches (Carduelis chloris and allies), Rhodospiza and Rhynchostruthus, and a well-supported clade with the aberrant Callacanthis and Pyrrhoplectes together with Carpodacus rubescens. Although part of the large Carduelis-Serinus complex, the poorly known Serinus estherae forms a distinct lineage without close relatives. The traditionally delimited genera Carduelis, Serinus, Carpodacus, Pinicola and Euphonia are polyphyletic or paraphyletic. Based on our results we propose a revised generic classification of finches and describe a new monotypic genus for Carpodacus rubescens.     

Diversity and species boundaries in floricolous downy mildews

Floricolous downy mildews are a monophyletic group of members of the genus Peronospora (Oomycota, Peronosporales). These downy mildews can be found on a variety of families of the Asteridae, including Asteraceae, Campanulaceae, Dipsacaceae, Lamiaceae, and Orobanchaceae. With the exception of Peronospora radii, which can also cause economically relevant losses, sporulation usually takes place only on floral parts of their hosts. However, only very few specimens of these mostly inconspicuous downy mildews have so far been included in molecular phylogenies. Focusing on Lamiaceae, we have investigated multiple specimens of floricolous downy mildews for elucidating species boundaries and host specificity in this group. Based on both mitochondrial and nuclear loci, it became apparent that phylogenetic lineages in the Lamiaceae seem to be host genus specific and significant sequence diversity could be found between lineages. Based on distinctiveness in both phylogenetic reconstructions and morphology, the downy mildew on flowers of Stachys palustris is introduced as a new species, Peronospora jagei sp. nov., which can be morphologically distinguished from Peronospora stigmaticola by broader and shorter conidiospores. The diversity of the floricolous down mildews might be higher than previously assumed, although specimens from a much broader set of samples will be needed to confirm this view.     

Pearl millet downy mildew as affected by integrating seed dressing,sowing date and cultivar

The effects of seed dressing, sowing date and cultivar on incidence and severity of downy mildew of pearl millet induced by Sclerospora graminicola and yield were studied in a two-year field trial conducted at the Research farm of University of Maiduguri. The millet cultivars, Ex-Borno, SOSAT-C88, GB 8735 and Gwagwa were each dressed with metalaxyl at 0.75 and 1.50 g a.i./kg seed; and a batch of undressed seeds of each cultivar served as control. Both dressed and undressed seeds were used for dry-planting and wet-planting in early and late seasons. The results showed that seed dressing with the fungicides significantly (p ≤ 0.05) reduced the incidence and severity of downy mildew and increased grain yield. Dry-planting also significantly (p ≤ 0.01) increased grain yield irrespective of disease incidence. Delay in sowing led to a significant reduction in incidence and severity of downy mildew. Differences between the cultivars in relation to incidence and severity of downy mildew and grain yield were significant. SOSAT-C88 developed low or no downy mildew in both seasons. Sowing of dressed SOSAT-C88 as soon as rainfall established appeared most beneficial in the control of downy mildew. Dry- or wet-planting Ex-Borno dressed with any of the metalaxyl formulations proved to be effective for downy mildew management and for high yield.     

Highly glycosylated flavonols at the genistoid boundary and the systematic position of Dermatophyllum

Among papilionoid legumes known to express the phenotype of quinolizidine alkaloid production, only Dermatophyllum occurs outside of the genistoid clade in phylogenetic analyses of DNA sequence data. Analysis of the foliar flavonoid glycosides of Dermatophyllum and possibly related clades, by liquid chromatography-UV spectrophotometry-mass spectrometry, revealed that taxa sampled from Dermatophyllum, Amphimas and the Cladrastis, lecointeoid and vataireoid clades contained mostly flavonol O-glycosides whereas taxa sampled from early-branching genistoid clades, the Andira clade and Aldina contained mostly flavone C-glycosides. Furthermore, leaves of Dermatophyllum secundiflorum and Dermatophyllum arizonicum contained, as their main flavonoids, two highly glycosylated flavonols: kaempferol 3-O-α-rhamnopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-galactopyranoside-7-O-α-rhamnopyranoside and its quercetin analogue. These compounds also occurred in Cladrastis kentukea, Styphnolobium japonicum and Pickeringia montana in the Cladrastis clade, Uribea tamarindoides and some samples of Zollernia in the lecointeoid clade, and in Amphimas pterocarpoides (another genus of uncertain relationships). The alkaloid and flavonoid phenotypes of Dermatophyllum each suggest affinities to different groups — a conflict which is accommodated by the current phylogenetic hypothesis, based on molecular data, that the genus is a possible sister to the genistoid clade but not a member of it.     

Control of pearl millet downy mildew caused by Sclerospora graminicola with systemic fungicides in an artificially-contaminated plot

Among four fungicides, viz. metalaxyl (two formulations), fosetyl-Al, pro-pamocarb and cyomaxanil tested in vitro against sporangial germination inhibition of Sclerospora graminicola, cyomaxanil was found to be most inhibitory. In an artificially contaminated plot, when used as seed treatment or foliar spray for the control of downy mildew of pearl millet, only metalaxyl was effective. Metalaxyl 25 (Ridomil) and metalaxyl 35 (Apron) seed treatments protected the pearl millet plants from downy mildew up to 30 days. As a foliar spray, metalaxyl 25 used once at 20 days or twice after 20 and 38 days of plant growth gave less disease at harvest time. Seed treatment (metalaxyl 25 or 35) followed by one metalaxyl 25 spray was found to be effective in controlling the downy mildew. These treatments improved the growth of plants and yield significantly.