The cellulose synthase 3 (CesA3) gene of oomycetes: structure, phylogeny and influence on sensitivity to carboxylic acid amide (CAA) fungicides

Proper disease control is very important to minimize yield losses caused by oomycetes in many crops. Today, oomycete control is partially achieved by breeding for resistance, but mainly by application of single-site mode of action fungicides including the carboxylic acid amides (CAAs). Despite having mostly specific targets, fungicidal activity can differ even in species belonging to the same phylum but the underlying mechanisms are often poorly understood. In an attempt to elucidate the phylogenetic basis and underlying molecular mechanism of sensitivity and tolerance to CAAs, the cellulose synthase 3 (CesA3) gene was isolated and characterized, encoding the target site of this fungicide class. The CesA3 gene was present in all 25 species included in this study representing the orders Albuginales, Leptomitales, Peronosporales, Pythiales, Rhipidiales and Saprolegniales, and based on phylogenetic analyses, enabled good resolution of all the different taxonomic orders. Sensitivity assays using the CAA fungicide mandipropamid (MPD) demonstrated that only species belonging to the Peronosporales were inhibited by the fungicide. Molecular data provided evidence, that the observed difference in sensitivity to CAAs between Peronosporales and CAA tolerant species is most likely caused by an inherent amino acid configuration at position 1109 in CesA3 possibly affecting fungicide binding. The present study not only succeeded in linking CAA sensitivity of various oomycetes to the inherent CesA3 target site configuration, but could also relate it to the broader phylogenetic context.     

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.     

A molecular phylogeny of Phytophthora and related oomycetes

Phylogenetic relationships among 50 Phytophthora species and between Phytophthora and other oomycetes were examined on the basis of the ITS sequences of genomic rDNA. Phytophthora grouped with Pythium, Peronospora, and Halophytophthora, distant from genera in the Saprolegniales. Albugo was intermediate between these two groups. Unlike Pythium, Phytophthora was essentially monophyletic, all but three species forming a cluster of eight clades. Two clades contained only species with nonpapillate sporangia. The other six clades included either papillate and semipapillate, or semipapillate and nonpapillate types, transcending traditional morphological groupings, which are evidently not natural assemblages. Peronospora was related to P. megakarya and P. palmivora and appears to be derived from a Phytophthora that has both lost the ability to produce zoospores and become an obligate biotroph. Three other Phytophthoras located some distance from the main Phytophthora-Peronospora cluster probably represent one or more additional genera.     

Occurrence,distribution and seasonality of aquatic fungi as affected by chemical factors in six alkaline ponds of Indi*

The periodicity of 30 species of water mould, belonging to the orders Blastocladiales, Saprolegniales, Lagenidiales, and Peronosporales, inhabiting six alkaline ponds near Lucknow, India, was found to be governed significantly by factors such as water temperature, dissolved oxygen and calcium.Part of the thesis approved by the University of Lucknow for the award of a Ph.D. degree.     

中国霜霉的几个新种

本文描述了霜霉属(Peronospora)两个新种。即:寄生于唇形科植物香薷(Elsholtziapatrnia Garcke)上的香薷霜霉(Peronospora elsholtziae sp.nov.)和寄生于大花益母草(Leonurusmacranthus Maxim.)上的大花益母草霜霉(Peronospora leonuri sp.nov);单轴霉属(Plasmopara)一个新种,寄生于车前草科植物车前草(Plantago major L.)上的车前草单轴霉(Plasmoparaplantaginicola sp.nov.,);盘梗霉属(Bremia)一个新种,寄生于石竹科植物莫石竹(Moehringialateriflora L.)上的莫石竹盘梗霉(Bremia moehringiae sp.nov.).这四个新种均有拉丁文和中文描述。还分别讨论了新种与同科近似种之间的区别。     

Occurrence of water molds in relation to hydrogen ion concentration in some lakes of Nainital,India

26 species of water molds belonging to Blastocladiales, Saprolegniales, Lagenidiales and Peronosporales were found to be different in their pH tolerance.     

Occurrence,distribution and seasonal periodicity of some aquatic fungi of Sat-Tal (Nainital), India

The occurrence of 23 species of aquatic fungi belonging to Blastocladiales, Saprolegniales, Lagenidiales and Peronosporales in three lakes and their surrounding cultivated and non-cultivated soils of Sat-Tal area have been studied. The species were found to be different in their habitat and temperature range.     

中国菊科植物上拟盘梗霉属的新种和新组合*

拟盘梗霉属(Bremiella G.W.Wilson)霜霉,全世界已报道2个种。本文报道寄生于我国菊科植物上的1个新种及2个新组合。新种是寄生于茼蒿上的Bremiella multiformis Tao et Y.Qin sp.nov.。新组合是欧茼蒿上的Bremiella chrysanthemi-coronarii(Sawada)Tao comb.nov。及黄花蒿上的Bremiella artemisiae-annuae(Ling et M. C.Tai)Tao comb.nov.。对新种作了汉文及拉丁文描述,并附有形态图,还讨论了新种和新组合与近似种之间的区别。     

中国菊科植物上拟盘梗霉属的新种和新组合

拟盘梗霉属(Bremiella G.W.Wilson)霜霉,全世界已报道2个种。本文报道寄生于我国菊科植物上的1个新种及2个新组合。新种是寄生于茼蒿上的Bremiella multiformis Tao et Y.Qin sp.nov.。新组合是欧茼蒿上的Bremiella chrysanthemi-coronarii(Sawada)Tao comb.nov。及黄花蒿上的Bremiella artemisiae-annuae(Ling et M. C.Tai)Tao comb.nov.。对新种作了汉文及拉丁文描述,并附有形态图,还讨论了新种和新组合与近似种之间的区别。     

Seasonal periodicity of aquatic fungi in tanks at Kurukshetra,India

The occurrence, distribution and seasonal periodicity of 49 species of aquatic fungi belonging to the orders Chytridiales, Lagenidiales, Blastocladiales, Saprolegniales and Peronosporales from two tanks of Kurukshetra, India, is described. Seasonal periodicity was found to be governed by temperature.     

Site‐specific distribution of oak rhizosphere‐associated oomycetes revealed by cytochrome c oxidase subunit II metabarcoding

The phylum Oomycota comprises important tree pathogens like Phytophthora quercina, involved in central European oak decline, and Phytophthora cinnamomi shown to affect holm oaks among many other hosts. Despite the importance to study the distribution, dispersal and niche partitioning of this phylum, metabarcoding surveys, and studies considering environmental factors that could explain oomycete community patterns are still rare. We investigated oomycetes in the rhizosphere of evergreen oaks in a Spanish oak woodland using metabarcoding based on Illumina sequencing of the taxonomic marker cytochrome c oxidase subunit II (cox2). We developed an approach amplifying a 333 bp long fragment using the forward primer Hud‐F (Mycologia, 2000) and a reverse primer found using DegePrime (Applied and Environmental Microbiology, 2014). Factors reflecting topo‐edaphic conditions and tree health were linked to oomycete community patterns. The majority of detected OTUs belonged to the Peronosporales. Most taxa were relatives of the Pythiaceae, but relatives of the Peronosporaceae and members of the Saprolegniales were also found. The most abundant OTUs were related to Globisporangium irregulare and P. cinnamomi, both displaying strong site‐specific patterns. Oomycete communities were strongly correlated with the environmental factors: altitude, crown foliation, slope and soil skeleton and soil nitrogen. Our findings illustrate the significance of small scale variation in habitat conditions for the distribution of oomycetes and highlight the importance to study oomycete communities in relation to such ecological patterns.     

The influence of site and position in the plant community on the nutrient distribution in,and content of Eichhornia crassipes (Mart.) Solms

The distribution of 26 species of Blastocladiales, Saprolegniales, Lagenidiales and Peronosporales from temperate and sub tropical lakes, situated at different altitude in the same locality, is described. Seasonal periodicity is treated and found to be governed by temperature.     

Phylogeny of Gardenieae (Rubiaceae) based on chloroplast DNA sequences from the rps16 intron and trnL(UAA)-F(GAA) intergenic spacer

Chloroplast DNA (cpDNA) sequences of the rps 16 intron and the trn L (UAA)-F(GAA) intergenic spacer were obtained from 59 species, to evaluate phylogenetic relationships among members of the Gardenieae, Rubiaceae. The results indicate that Gardenieae is polyphyletic and that genera included in the subtribe Diplosporinae should be transferred to the Coffeeae and Octotropideae. The Octotropideae tribe may also be polyphyletic. It is also demonstrated that the tribe Pavetteae is polyphyletic. In addition, the present data support a basal position for the disputed genus Bertiera within the Coffeeae clade. Of the informal groups proposed by Robbrecht & Puff within the Gardenieae, the Alibertia group is strongly supported, and is estimated to also include Ibetralia, Genipa aff. williamsii , and one undescribed taxon. However, Genipa americana was not part of this clade suggesting that Genipa is polyphyletic. The Tetrad group is not supported, and character optimisations suggest that pollen released in tetrads may have arisen three times. Most of these genera appear in the same clade as the three neotropical genera Sphinctanthus, Rosenbergiodendron , and Tocoyena , all of which bear pollen in monads. Atractogyne, Mitriostigma , and Oxyanthus form the second group with pollen in tetrads, whereas Gardenia is embedded in a third group. Massularia , with polyads, occurs either singly on a large polytomy or at the base of the Atractocarpus-Porterandia-Trukia clade.     

Cryptolepiota, a new sequestrate genus in the Agaricaceae with evidence for adaptive radiation in western North America

Phylogenetic analyses based on nLSU and ITS sequence data indicate that the sequestrate genus Gigasperma is polyphyletic. Gigasperma cryptica, which is known only from New Zealand, has affinities with the Cortinariaceae whereas G. americanum and two additional undescribed taxa from western North America are derived from Lepiota within the Agaricaceae. The three North American taxa appear to be recently evolved and are closely related. They occur in similar environments and form a well supported clade indicating that adaptive radiation has occurred within this group of fungi. An independent genus with sequestrate fructifications, Cryptolepiota is proposed to accommodate the three species in this clade. Cryptolepiota microspora and C. mengei are described as new, and G. americanum is transferred to Cryptolepiota. Gigasperma cryptica is illustrated and compared with the species of Cryptolepiota.     

Tobacco blue mould disease caused by Peronospora hyoscyami f. sp. tabacina

Blue mould [ Peronospora hyoscyami f. sp. tabacina (Adam) Skalicky 1964] is one of the most important foliar diseases of tobacco that causes significant losses in the Americas, south-eastern Europe and the Middle East. This review summarizes the current knowledge of the mechanisms employed by this oomycete pathogen to colonize its host, with emphasis on molecular aspects of pathogenicity. In addition, key biochemical and molecular mechanisms involved in tobacco resistance to blue mould are discussed.
Taxonomy: Kingdom: Chromista (Straminipila); Phylum: Heterokontophyta; Class: Oomycete; Order: Peronosporales; Family: Peronosporaceae; Genus: Peronospora ; Species: Peronospora hyoscyami f. sp. tabacina .
Disease symptoms: The pathogen typically causes localized lesions on tobacco leaves that appear as single, or groups of, yellow spots that often coalesce to form light-brown necrotic areas. Some of the leaves exhibit grey to bluish downy mould on their lower surfaces. Diseased leaves can become twisted, such that the lower surfaces turn upwards. In such cases, the bluish colour of the diseased plants becomes quite conspicuous, especially under moist conditions when sporulation is abundant. Hence the name of the disease: tobacco blue mould.
Infection process: The pathogen develops haustoria within plant cells that are thought to establish the transfer of nutrients from the host cell, and may also act in the delivery of effector proteins during infection.
Resistance: Several defence responses have been reported to occur in the Nicotiana tabacum – P. hyoscyami f. sp. tabacina interaction. These include the induction of pathogenesis-related genes, and a correlated increase in the activities of typical pathogenesis-related proteins, such as peroxidases, chitinases, β-1,3-glucanases and lipoxygenases. Systemic acquired resistance is one of the best characterized tobacco defence responses activated on pathogen infection.     

Sterol biosynthesis in oomycete pathogens

Oomycetes are a diverse group of filamentous eukaryotic microbes comprising devastating animal and plant pathogens. They share many characteristics with fungi, including polarized hyphal extension and production of spores, but phylogenetics studies have clearly placed oomycetes outside the fungal kingdom, in the kingdom Stramenopila which also includes marine organisms such as diatoms and brown algae. Oomycetes display various specific biochemical features, including sterol metabolism. Sterols are essential isoprenoid compounds involved in membrane function and hormone signaling. Oomycetes belonging to Peronosporales, such as Phytophthora sp., are unable to synthesize their own sterols and must acquire them from their plant or animal hosts. In contrast, a combination of biochemical and molecular approaches allowed us to decipher a nearly complete sterol biosynthetic pathway leading to fucosterol in the legume pathogen Aphanomyces euteiches, an oomycete belonging to Saprolegniales. Importantly, sterol demethylase, a key enzyme from this pathway, is susceptible to chemicals widely used in agriculture and medicine as antifungal drugs, suggesting that similar products could be used against plant and animal diseases caused by Saprolegniales.Key words: azoles, fungicides, root rot, elicitin, Saprolegnia, chromoalveolates     

Ecological studies of some aquatic phycomycetes

Summary A survey of the phycomycete flora of three types of collecting stations in Allahabad was made for a year. Twelve species were recorded, belonging to the Saprolegniales, Blastocladiales and Peronosporales. The results indicate that the rarity of many of the aquatic phycomycetes may be due not only to specificity of substratum but also to temperature and the time needed for maturation of the oospores. There is a distinct seasonal periodicity of growth which is mainly restricted to winter months.     

疫霉属菌种的液氮超低温保藏

对疫霉属的15个种68株菌和霜疫霉2株菌进行了液氮超低温的保藏试验并得到成功。比较了冷冻速度、保护剂和解冻速度对菌存活的影响。严格控制每分钟降温摄氏1度直到-40℃后再放入液氮罐中,对疫霉和霜疫霉来说都是非常必要的。这种降温程序可通过简单设备人工操作达到。而直接由室温降到-150℃以下会损伤菌种以致死亡。在所用的保护剂中,不论10％甘油还是5-15％二甲基亚砜都能起到保护作用。尚看不出对那种保护剂有特别的要求,似可任意选用。至于解冻条件,由液氮中取出放置在38℃水浴中快速融化与在20℃水浴中中等速度融化效果相等,对菌的成活都没有太大影响。我们的试验肯定了在有保护剂存在下,用慢速冷冻可以在液氮中保存疫霉和霜疫霉。     

Mitochondrial and Nuclear DNA Restriction Enzyme Analysis of the Closely Related Phytophthora Species P. infestans, P. mirabilis, and P. phaseoli

To determine relatedness of the phytopathogenic fungi Phytophthora infestans , P. mirabilis , and P. phaseoli restriction fragment patterns of mitochondrial DNAs of several isolates and hybridization patterns of nuclear DNAs after Southern hybridization with a specific homologous probe were analyzed.
All but two isolates of P. infestans and P. mirabilis show very similar restriction fragment patterns differing only in the length of one fragment due to small insertion/deletion(s). Two isolates of P. mirabilis have one additional site for Scr FI. On the contrary at least six sites differ in P. phaseoli when compared to the other two species. The mitochondrial genome of P. phaseoli is considerably smaller (approx. 6 kbp) than those of P. infestans and P. mirabilis .
A cloned 430 bp multicopy DNA sequence, derived from P. infestans , hybridized specifically with P. infestans, P. mirabilis , and P. phaseoli out of 61 species of Peronosporales ( Phytophthora, Halophytophthora, Pythium, Albugo, Bremia, Peronospora, Plasmopara ) tested and therefore has potential as a diagnostic probe. Restriction patterns revealed by this probe are invariant intraspecifi-cally but differ between the three species.
We consider P. mirabilis a forma specialis of P. infestans because of the very high similarly in its mitochondrial DNA restriction patterns.     

基于rDNA ITS序列探讨部分腐霉种的系统发育与其形态特征

基于对73株计58种腐霉和6种疫霉的核糖体DNA的ITS序列分析,以海生疫霉为外围群,按邻接法构建系统发育树,对腐霉的系统发育关系进行了研究。结果表明:在58种腐霉中,Pythium ostracodes,P.chamaehyphon,P.carbonicum,P.montanum和P.vexans归为同一组,介于其它腐霉和疫霉之间,这5种腐霉的孢子囊均为球形;现已归为疫霉属的P.undulatum 单独为一组,它与腐霉的亲缘关系比疫霉更近;其余52种腐霉聚成一大组,这52种腐霉基本上按孢子囊或菌丝膨大体形态分成Ⅰ、Ⅱ两组:第1组31种腐霉, 其中30种腐霉的孢子囊或菌丝膨大体为球形;第Ⅱ组21种腐霉,其中19种腐霉的孢子囊为丝状、瓣状或裂片状。基于ITS序列分析,腐霉属的其它性状如藏卵器壁是否光滑、卵孢子是否满器、雄器的着生方式和数量、异宗配合等呈多元演化。