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.     

Re-consideration of Peronospora farinosa infecting Spinacia oleracea as distinct species, Peronospora effusa

Downy mildew is probably the most widespread and potentially destructive global disease of spinach (Spinacia oleracea). The causal agent of downy mildew disease on various plants of Chenopodiaceae, including spinach, is regarded as a single species, Peronospora farinosa. In the present study, the ITS rDNA sequence and morphological data demonstrated that P. farinosa from S. oleracea is distinct from downy mildew of other chenopodiaceous hosts. Fifty-eight spinach specimens were collected or loaned from 17 countries of Asia, Europe, Oceania, North and South America, which all formed a distinct monophyletic group. No intercontinental genetic variation of the ITS rDNA within Peronospora accessions causing spinach downy mildew disease was found. Phylogenetic trees supported recognition of Peronospora from spinach as a separate species. Microscopic examination also revealed morphological differences between Peronospora specimens from Spinacia and P. farinosa s. lat. specimens from Atriplex, Bassia, Beta, and Chenopodium. Consequently, the name Peronospora effusa should be reinstated for the downy mildew fungus found on spinach. Here, a specimen of the original collections of Peronospora effusa is designated as lectotype.     

南美蟛蜞菊霜霉病病原鉴定及系统发育分析

[目的]为从天敌病原生物方面探索外来入侵植物南美蟛蜞菊的生物防治,对新发现的南美蟛蜞菊霜霉病进行病原鉴定和系统发育分析。[方法]在广东省广州市对南美蟛蜞菊霜霉病的发生及危害情况进行调查,并通过病害症状识别、病原显微形态记录与比较、病原菌及其近似种ITS序列结构比较、LSU序列和ITS序列系统发育分析,对南美蟛蜞菊霜霉病病原进行鉴定和系统发育分析。[结果]南美蟛蜞菊霜霉病在广州零星发生,但该病害在华南农业大学校园内发生较严重,发病率达50%~70%,病情指数为30~35。经鉴定,其病原菌为南美蟛蜞菊单轴霉,是国内一新记录种。基于病原菌LSU序列和ITS序列的系统发育分析显示,侵染菊科植物的单轴霉属菌种聚在一个分枝,亲缘关系密切,与侵染其他不同科寄主植物的单轴霉亲缘关系较远。ITS序列结构比较显示,寄生于菊科向日葵族植物的单轴霉属菌种的ITS2区包含多个重复序列,不同菌种间的ITS2区重复序列相似度不同,说明侵染菊科向日葵簇植物的单轴霉属菌物可细分成多个种,而不是只有向日葵单轴霉。[结论]广州发生的南美蟛蜞菊霜霉病是该寄主上首次正式报道的病害,鉴定的病原菌也是国内一新记录种;寄生在菊科植物上的单轴霉属种类不尽相同,但亲缘关系紧密。     

Phylogeny of <Emphasis Type="Italic">Hyaloperonospora</Emphasis> based on nuclear ribosomal internal transcribed spacer sequences

Phylogenetic relationships in Hyaloperonospora (Oomycetes) were investigated by molecular analyses using internal transcribed spacer (ITS) sequences and collections from different host plants. Trees were inferred with Bayesian Markov chain Monte Carlo, neighbor-joining and maximum parsimony methods and rooted with Perofascia. The results are discussed with respect to host taxonomy and species concepts of downy mildews from the literature. Molecular data mainly support the use of narrow species delimitations and host range as a taxonomic marker. Hyaloperonospora brassicae turns out to be a non-monophyletic assemblage of different species. New combinations are proposed in accordance with the phylogenetic trees.     

Internal transcribed spacer 2 amplicon as a molecular marker for identification of Peronospora parasitica (crucifer downy mildew)

AIMS: The purpose of the study was to characterize the internal transcribed spacer (ITS) regions of Peronospora parasitica (crucifer downy mildew) in order to evaluate their potential as molecular markers for pathogen identification. METHODS AND RESULTS: PCR amplification of ribosomal RNA gene block (rDNA) spacers (ITS1 and ITS2) performed in 44 P. parasitica isolates from different Brassica oleracea cultivars and distinct geographic origins, revealed no length polymorphisms. ITS restriction analysis with three endonucleases, confirmed by sequencing, showed no fragment length polymorphisms among isolates. Furthermore, ITS amplification with DNA isolated from infected host tissues also allowed the detection of the fungus in incompatible interactions. The combination of the universal ITS4 and ITS5 primers, for amplification of full ITS, with a new specific forward internal primer for ITS2 (PpITS2F), originates a P. parasitica specific amplicon, suitable for diagnosis. CONCLUSIONS: As ITS2 regions of P. parasitica, B. oleracea, other B. oleracea fungal pathogens and other Peronospora species are clearly distinct, a fast and reliable molecular identification method based on multiplex PCR amplification of full ITS and P. parasitica ITS2 is proposed for the diagnosis of crucifer downy mildew. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be applied to diagnose the disease in the absence of fungal reproductive structures, thus being useful to detect nonsporulating interactions, early stages of infection on seedlings, and infected young leaves packed in sealed plastic bags. Screening of seed stocks in sanitary control is also a major application of this diagnostic method.     

Phylogenetic reassessment of the Teloschistaceae (lichen-forming Ascomycota, Lecanoromycetes)

The Teloschistaceae is a widespread family with considerable morphological and ecological heterogeneity across genera and species groups. In order to provide a comprehensive molecular phylogeny for this family, phylogenetic analyses were carried out on sequences from the nuclear ribosomal ITS region obtained from 114 individuals that represent virtually all main lineages of Teloschistaceae. Our study confirmed the polyphyly of Caloplaca, Fulgensia and Xanthoria, and revealed that Teloschistes is probably non-monophyletic. We also confirm here that species traditionally included in Caloplaca subgenus Gasparrinia do not form a monophyletic entity. Caloplaca aurantia, C. carphinea and C. saxicola s. str. groups were recovered as monophyletic. The subgenera Caloplaca and Pyrenodesmia were also polyphyletic. In the subgenus Caloplaca, the traditionally recognized C. cerina group was recovered as monophyletic. Because this study is based solely on ITS, to maximize taxon sampling, the inclusion of phylogenetic signal from ambiguously aligned regions in MP (recoded INAASE and arc characters) resulted in the most highly supported phylogenetic reconstruction, compared with Bayesian inference restricted to alignable sites.     

External flavonoid aglycones of Viguiera series Viguiera (asteraceae: heliantheae)

Distribution of external flavonoid aglycones supports other data to suggest that Viguiera series Viguiera (series Dentatae Blake) is a mixed taxonomic grouping. Two species, V. dentata and V. potosina, lack external flavonoid aglycones, whereas the remaining species of the series all produce external flavonoid aglycones and appear to form a natural taxonomic group. The latter group, which consists of species from Baja California and nearby xeric areas, exhibits a diversity of flavonoid types, with different diploid species variously producing flavonols, simple flavones, 6-methoxyflavones, 6-hydroxy,7-methoxyflavones and a chalkone as major compounds. Polyploid species exhibit combinations of flavonoid types from possible diploid ancestors. Compounds of the 6-hydroxy,7-methoxyflavone type appear to characterize the group of species from Baja California and nearby areas within Viguiera.     

Relationships between four novel ceratomyxid parasites from the gall bladders of labrid fishes from Heron Island, Queensland, Australia

Ceratomyxid parasites from the gall bladder of fishes from the family Labridae were examined for their taxonomic identity and their relatedness to other species of myxozoans. This paper describes four novel species Ceratomyxa choerodonae n. sp. from Choerodon cyanodus; Ceratomyxa cheilinae n. sp. from Cheilinus chlorourus; Ceratomyxa oxycheilinae n. sp. from Oxycheilinus digramma; and Ceratomyxa thalassomae n. sp. from Thalassoma lunare. Each species is characterised morphologically and small subunit (18 S) rDNA sequences were used in molecular phylogenetic analyses. Comparison of the small subunit rDNA sequences revealed four distinct species even though morphological differences indicated the presence of only two morphologically distinctive spore types. Phylogenetic analyses revealed that the ceratomyxids from labrid fishes did not form a clade to the exclusion of all other myxosporeans and provides evidence that host relatedness is not a good character to discriminate between species within the genus Ceratomyxa.     

Monophyly and systematic position of Glypthelmins (Digenea), based on partial lsrDNA sequences and morphological evidence

Species composition and systematic placement within the order Plagiorchiida has been controversial. Species number in Glypthelmins Stafford, 1905, a genus of cosmopolitan parasites of anurans, has varied between 19 and 28 species, depending on the taxonomic treatment. The present study performs a phylogenetic analysis using partial lsrDNA sequences to test the monophyly of the genus, and compares new sequences obtained with those published for different plagiorchiids to clarify the systematic position of Glypthelmins within the order Plagiorchiida. Maximum parsimony (MP) and maximum likelihood (ML) analyses result in identical tree topology. The single MP tree (L=1587, CI=0.40, RI=0.76) includes several clades with high bootstrap and Bremer support values. Glypthelmins sensu lato as traditionally classified is paraphyletic. Based on molecular and/or morphological evidence, the taxonomic diagnosis for Glypthelmins is emended, only eight species are retained in the genus, and re-establishment of the genera Choledocystus Pereira & Cuocolo, 1941 and Rauschiella Babero, 1951 is proposed, resulting in the following new combinations: Choledocystus simulans (Teixeira de Freitas, 1941) comb. nov., C. vitellinophilum (Dobbin, 1958) comb. nov.; Rauschiella chaquensis (Mañé-Garzón & Holcman-Spector, 1967) comb. nov., R. lenti (Teixeira de Freitas, 1941) comb. nov., R. linguatula (Rudolphi, 1819) comb. nov., R. poncedeleoni (Razo-Mendivil & León-Règagnon, 2001) comb. nov., R. robusta (Brooks, 1976) comb. nov., R. rugocaudata (Yoshida, 1916) comb. nov., R. staffordi (Tubangui, 1928) comb. nov. In the phylogenetic reconstruction, Glypthelmins sensu stricto forms the sister group of Haematoloechus Looss, 1899.     

Species of Colletotrichum on Agavaceae

Species of Colletotrichum cause diseases on a wide range of hosts, frequently infecting plants in the Agavaceae (monocotyledons: Liliales). Three species of Colletotrichum restricted to the Agavaceae were detected through morphological studies of specimens and molecular sequence analyses of the LSU of the nu-rDNA and the ITS region of the nu-rDNA from cultures. Colletotrichum agaves on Agave is fully described and illustrated. Colletotrichum dracaenophilum is described as a new species for isolates having long conidia and occurring on Dracaena sanderiana from China. Colletotrichum phormii and Glomerella phormii are determined to be the correct scientific names for the asexual and sexual states, respectively, of a species commonly referred to as C. rhodocyclum and G. phacidiomorpha occurring mainly on Phormium. In addition, C. gloeosporioides and C. boninense were isolated from plants in the Agavaceae. All species of Colletotrichum described on Agavaceae were evaluated based on type specimens. A key to the five species of Colletotrichum on Agavaceae is included. This paper includes one new species, Colletotrichum dracaenophilum, and three new combinations, Colletotrichum phormii, Glomerella phormii, and Phaeosphaeriopsis phacidiomorpha.     

Molecular Taxonomy of Phytopathogenic Fungi: A Case Study in Peronospora

Background

Inappropriate taxon definitions may have severe consequences in many areas. For instance, biologically sensible species delimitation of plant pathogens is crucial for measures such as plant protection or biological control and for comparative studies involving model organisms. However, delimiting species is challenging in the case of organisms for which often only molecular data are available, such as prokaryotes, fungi, and many unicellular eukaryotes. Even in the case of organisms with well-established morphological characteristics, molecular taxonomy is often necessary to emend current taxonomic concepts and to analyze DNA sequences directly sampled from the environment. Typically, for this purpose clustering approaches to delineate molecular operational taxonomic units have been applied using arbitrary choices regarding the distance threshold values, and the clustering algorithms.

Methodology

Here, we report on a clustering optimization method to establish a molecular taxonomy of Peronospora based on ITS nrDNA sequences. Peronospora is the largest genus within the downy mildews, which are obligate parasites of higher plants, and includes various economically important pathogens. The method determines the distance function and clustering setting that result in an optimal agreement with selected reference data. Optimization was based on both taxonomy-based and host-based reference information, yielding the same outcome. Resampling and permutation methods indicate that the method is robust regarding taxon sampling and errors in the reference data. Tests with newly obtained ITS sequences demonstrate the use of the re-classified dataset in molecular identification of downy mildews.

Conclusions

A corrected taxonomy is provided for all Peronospora ITS sequences contained in public databases. Clustering optimization appears to be broadly applicable in automated, sequence-based taxonomy. The method connects traditional and modern taxonomic disciplines by specifically addressing the issue of how to optimally account for both traditional species concepts and genetic divergence.     

内生枯草芽孢杆菌JL4在葡萄叶上的定殖及其对葡萄霜霉病的防治

为了明确枯草芽孢杆菌JL4在葡萄叶表面和内部的定殖情况,研究定殖与防治效果的关系,采用电击转化的方法将含有GFP基因的质粒pGFP78导入枯草芽孢杆菌JL4中,并得到成功表达GFP 的生防菌JL4-gfp,测试了标记菌株的稳定性及其对葡萄霜霉病菌的抑制作用.采用叶片喷雾法接种,用抗生素平板稀释分离回收,检测生防菌JL4-gfp在葡萄叶片的定殖情况,并将采回的叶片在室内接种葡萄霜霉菌孢子囊悬浮液进行生防测定.结果表明: 标记菌株在经过10次传代培养后,仍具有良好的发光表型,能稳定表达GFP蛋白,并且标记菌株JL4-gfp对葡萄霜霉菌保持了原有的抑菌作用;用抗生素平板稀释分离回收,检测到JL4-gfp菌株在葡萄叶片表面的定殖量在接种后的0、3和7 d分别为3.6×10⁵、2.7×10⁵和3.1×10³ CFU·g^-1;叶片内部的定殖在接种3 d后达到最大(9.6×10⁴ CFU·g^-1),然后下降,14 d后已经检测不到接种菌株;室内生防测定结果显示,喷雾后3 d对葡萄霜霉病的防治效果达88.0%以上,但7 d后则无明显防效.JL4-gfp的定殖量与其防治葡萄霜霉病的效果呈正相关,其有效定殖量临界值为10⁵ CFU·g^-1.     

Morphological and Molecular Characterization of the Causal Agent of Downy Mildew on Quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis>)

Downy mildew is an economically important and widespread disease in quinoa (Chenopodium quinoa) growing areas. Although in many studies Peronospora farinosa is most commonly regarded as the causal agent of the disease, identification and classification of the pathogen remain still uncertain due to its taxonomic confusion. Thirty-six Peronospora isolates from quinoa with different geographic origins including Argentina, Bolivia, Denmark, Ecuador, and Peru were morphologically and molecularly compared with Peronospora species from other Chenopodium species. The morphology of three herbarium specimens was similar to that of P. variabilis, which originated from C. album, characterized by flexuous to curved ultimate branchlets and pedicellated conidia. Phylogenetic analysis based on ITS rDNA sequences also placed the quinoa pathogen within the same clade as P. variabilis. Within the ITS rDNA sequences of the quinoa pathogens, two base substitutions were found, which separated the majority of the Danish isolates from isolates from South America, but no sequence difference was found among the isolates from different cultivars of quinoa. The present results indicate that the pathogen responsible for the quinoa downy mildew is identical to Peronospora variabilis and that it should not be lumped with P. farinosa as claimed previously by most studies.     

根虫瘟霉和相关种的分类地位研究及其ITS核苷酸序列分析

根虫瘟霉是最常见的一种虫霉,寄主广泛,世界广布。目前有学者认为该种是个复合种。本研究对世界不同地区和不同寄主的根虫瘟霉及其近缘类群总计19个菌株,进行3个靶位点（ITS、LSU rDNA、RPB2）的分子系统发育学分析。结果显示,根虫瘟霉ITS长度较为保守性,介于1 321-1 324bp之间,而所研究的虫霉亚门的其他类群的长度范围较大,为556-1 654bp。本研究确认根虫瘟霉是单系种,同时西虫瘟霉、矛孢虫瘟霉和英吉利虫瘟霉具有明确种的分类地位。鬼笔状虫瘟霉种应该被视为西虫瘟霉的异名。     

Erysiphe abbreviata on cherry bark oak--morphology, phylogeny and taxonomy

Powdery mildew on cherry bark oak (Quercus falcate var. pagodifolia) collected in Tennessee, USA, was determined to be Erysiphe abbreviata, a species confined to North America. The diagnostically important anamorph of this species is described for the first time. Sequence analyses of the rDNA ITS region and D1/D2 domains of the 28S rDNA were used to obtain phylogenetic data for and taxonomic conclusions about this species. The structure of the anamorph (Oidium subgen. Pseudoidium) and the molecular data support the placement of this species in Erysiphe emend. (including Microsphaera) as a species separate from the Eurasian Erysiphe alphitoides.     

Antechiniscus in Australia: Description of Antechiniscus moscali sp. n. and Redescription of Antechiniscus parvisentus (Horning & Schuster, 1983) (Heterotardigrada: Echiniscidae)

Two species of the genus Antechiniscus have been found in Nothofagus forests in Australia. The new species A. moscali sp. n. is described. The New Zealand species A. parvisentus (Horning & Schuster, 1983) is redescribed from type material and from the new material from Australia. The taxonomic status of the two species within the genus is discussed.     

How do obligate parasites evolve? A multi-gene phylogenetic analysis of downy mildews

Plant parasitism has independently evolved as a nutrition strategy in both true fungi and Oomycetes (stramenopiles). A large number of species within phytopathogenic Oomycetes, the so-called downy mildews, are defined as obligate biotrophs since they have not, to date, been cultured on any artificial medium. Other genera like Phytophthora and Pythium can in general be cultured on standard or non-standard agar media. Within all three groups there are many important plant pathogens responsible for severe economic losses as well as damage to natural ecosystems. Although they are important model systems to elucidate the evolution of obligate parasites, the phylogenetic relationships between these genera have not been clearly resolved. Based on the most comprehensive sampling of downy mildew genera to date and a representative sample of Phytophthora subgroups, we inferred the phylogenetic relationships from a multi-gene dataset containing both coding and non-coding nuclear and mitochondrial loci. Phylogenetic analyses were conducted under several optimality criteria and the results were largely consistent between all the methods applied. Strong support is achieved for monophyly of a clade comprising both the genus Phytophthora and the obligate biotrophic species. The facultatively parasitic genus Phytophthora is shown to be at least partly paraphyletic. Monophyly of a cluster nested within Phytophthora containing all obligate parasites is strongly supported. Within the obligate biotrophic downy mildews, four morphologically or ecologically well-defined subgroups receive statistical support: (1) A cluster containing all species with brownish-violet conidiosporangia, i.e., the genera Peronospora and Pseudoperonospora; (2) a clade comprising the genera with vesicular to pyriform haustoria (Basidiophora, Benua, Bremia, Paraperonospora, Plasmopara, Plasmoverna, Protobremia); (3) a group containing species included in Hyaloperonospora and Perofascia which almost exclusively infect Brassicaceae; (4) a clade including the grass parasites Viennotia oplismeni and Graminivora graminicola. Phylogenetic relationships between these four clades are not clearly resolved, and neither is the position of Sclerospora graminicola within the downy mildews. Character analysis indicates an evolutionary scenario of gradually increasing adaptation to plant parasitism in Peronosporales and that at least the most important of these adaptive steps occurred only once, including major host shifts within downy mildews.     

高茎紫菀(菊科)形态描述补充及细胞学和分子系统学研究北大核心CSCD

结合在模式产地采集的高茎紫菀(Aster procerus Hemsley)以及查阅文献资料,发现原始文献对其形态描述不充分且缺乏细胞学和分子系统学方面的报道。本研究对高茎紫菀的形态特征进行了补充描述以及核型特征和系统位置分析,为紫菀属的修订提供资料。结果表明:(1)依据观察结果,补充了高茎紫菀新的形态特征:基生叶羽状分裂,成熟的基生叶较大,长可达26 cm,宽可达8 cm;花序托圆锥状。(2)高茎紫菀的染色体数目为2n=18;核型公式为2n=2x=16 m+2 M,核型属于1 A。(3)基于ITS和ETS标记的分子系统发育树分析表明,高茎紫菀不同居群的2个个体在同一进化支上(LP=100,PP=1.00),且位于核心紫菀属(LP=100,PP=1.00),与女菀[Turczaninovia fastigiata(Fischer)Candolle]构成姐妹类群(LP=52,PP=0.99)。研究认为,高茎紫菀基生叶和花序托的特征可为紫菀属的分类提供新的证据,支持高茎紫菀位于紫菀属(Aster L.)内,建议将女菀并入紫菀属。