Safflower Seedling a Selective Host to Discriminate Phytophthora melonis from Phytophthora drechsleri

Safflower seedlings were used to discriminate two morphologically similar species of Phytophthora namely P. melonis and P. drechsleri . All isolates of P. melonis from different sources could not infect safflower seedlings under high inoculum potential whereas all isolates of P. drechsleri from various hosts attacked safflower within a short period. Of 31 authentic Phytophthora species inoculated to safflower seedlings only seven species including Phytophthora asparagi , Phytophthora cactorum , Phytophthora cryptogea , Phytophthora drechsleri , Phytophthora erythroseptica , Phytophthora palmivora and Phytophthora quercina caused hypocotyls infection. All cucurbit isolates of Phytophthora from different parts of Iran and one from China could not infect safflower seedlings and were identified as P. melonis which had been confirmed previously by molecular analysis.     

Single-strand-conformation polymorphism of ribosomal DNA for rapid species differentiation in genus Phytophthora

Single-strand-conformation polymorphism (SSCP) of ribosomal DNA of 29 species (282 isolates) of Phytophthora was characterized in this study. Phytophthora boehmeriae, Phytophthora botryosa, Phytophthora cactorum, Phytophthora cambivora, Phytophthora capsici, Phytophthora cinnamomi, Phytophthora colocasiae, Phytophthora fragariae, Phytophthora heveae, Phytophthora hibernalis, Phytophthora ilicis, Phytophthora infestans, Phytophthora katsurae, Phytophthora lateralis, Phytophthora meadii, Phytophthora medicaginis, Phytophthora megakarya, Phytophthora nicotianae, Phytophthora palmivora, Phytophthora phaseoli, Phytophthora pseudotsugae, Phytophthora sojae, Phytophthora syringae, and Phytophthora tropicalis each showed a unique SSCP pattern. Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora drechsleri, and Phytophthora megasperma each had more than one distinct pattern. A single-stranded DNA ladder also was developed, which facilitates comparison of SSCP patterns within and between gels. With a single DNA fingerprint, 277 isolates of Phytophthora recovered from irrigation water and plant tissues in Virginia were all correctly identified into eight species at substantially reduced time, labor, and cost. The SSCP analysis presented in this work will aid in studies on taxonomy, genetics, and ecology of the genus Phytophthora.     

福建、浙江、江苏、上海疫霉种的研究

1985—1986年,从福建漳州,浙江杭州,上海,江苏南京,苏州,扬州,常州等地采集100多种植物进行分离,在雪松(Cedrus deodara),倒挂仙人鞭(Cereus flagelliformis),冬青卫茅(Euonymus japonieus),构树(Broussonetia papyrifera),蓖麻(Ricinus communis),柑桔(Citrus reticulata),带叶兜兰(Paphiopedilum hirsutissimum),芋(Colocasia esculenta),鳄梨(Persea americana),山茶(Camellia sp.),珊瑚樱(Solanum pseudo-capsicum),百合(Lilium brownii var.viridulum),蔓常春花(Vinca major),西瓜(Citrullus lanatus)等14种寄主植物上分离到51个疫霉菌株,鉴定为9个种:烟草疫霉(=寄生疫霉)Phytophthora nicotianae B.de Haan(=P.parasitica Dast.),掘氏疫霉P.drechsleri Tucker,樟疫霉 P.ciunamomi Rands,棕榈疫霉P.palmivora(Butl.)Butl.,柑桔褐腐疫霉P.citrophthora(R.et E.Smith) Leon.,苎麻疫霉P.bochmeriae Saw.,蜜色疫霉P.meadii McRae,簇囊疫霉P.botryosa Chee,隐地疫霉P.cryptogea Pethyb.et Laff.。其中,烟草疫霉,掘氏疫霉,樟疫霉出现频率较高,分布较广,初步认为是上述地区植物疫病的主要病原菌。簇囊疫霉、蜜色疫霉是国内新记录。     

Zoospore chemotaxis in Australian isolates of Phytophthora species.

Zoospores of Australian isolates of Phytophthora drechsleri, P. cryptogea, P. cinnamomi, P. nicotianae var. parasitica, and P. citricola were examined for their chemotactic responses to asparagine, glutamine, aspartate, glutamate, and structurally related compounds. Structural requirements for attraction include the alpha-amino-acid group with a short carbon chain terminating in an amide group. The one American isolate tested gave a different result and possible reasons for this are discussed. The pH of the environment was important, a neutral-charged molecule was more attractive than a negatively charged molecule, hence glutamine and aspartate were more attractive at pH 3.0 than pH 5.0. Zoospores tended to move away from regions with a high hydrogen ion concentration. Compounds other than amino acids were slightly attractive including several sugars and ethanol. Synergistic interactions between amino acids, ethanol, and sucrose were observed and may account for the high levels of attraction of zoospores to root exudates and extracts.     

Aminopeptidase profiles of four species of phytophthora

Phytophthora palmivora, P. cinnamomi, P. drechsleri, and P. cactorum were readily separated on the basis of the aminopeptidase substrate specificities of their mycelial suspensions. Variability among isolates of the same species was not significant with most substrates and isolates tested, regardless of source. Both qualitative and quantitative differences in enzyme activity were useful for species identification. Differentiation of these four species was possible with comparative reactions of l-alanyl-, l-arginyl-, l-benzoylarginyl, l-gamma-glutamyl-, l-glycyl-, l-hydroxyprolyl-, l-leucyl-, l-lysyl-, 4-methoxyleucyl-, l-prolyl-, and 4-methoxyalanyl-beta-naphthylamides. Variation in peptidase activity was usually detectable after 4 h of incubation, with increased activity sometimes manifest after 24 h of incubation. P. palmivora exhibited the lowest, and P. drechsleri exhibited the highest, overall peptidase activity. This fluorescent aminopeptidase profile procedure provides a relatively rapid method to supplement other taxonomic criteria for identification of these species of Phytophthora.     

六种疫霉菌体可溶性蛋白质和酯酶的聚丙烯酰胺凝胶平板电泳研究*

本文对槐生疫霉(Phytophthora robinicola)、掘氏疫霉(P.drechsleri)、樟疫霉(P.cinnamomi)、烟草疫霉(P.nicotianae)、棕榈疫霉(P.palmivora)及辣椒疫霉(P.capsici)等6种疫霉81株菌菌体可溶性蛋白质和酯酶进行了聚丙烯酰胺凝胶平板电泳研究。结果指出,种间蛋白质图谱差异明显,种内菌株间基本一致。就酯酶图谱来说,种内菌株间存在一定差异,但种间差异更为显著。因此,本试验进一步支持槐生疫霉新种的建立。同时表明,菌体可溶性蛋白质电泳图谱在疫霉种的鉴定和分类上具有重要意义,而酯酶电泳图谱在一定程度上也有助于疫霉种的研究。     

六种疫霉菌体可溶性蛋白质和酯酶的聚丙烯酰胺凝胶平板电泳研究

本文对槐生疫霉(Phytophthora robinicola)、掘氏疫霉(P.drechsleri)、樟疫霉(P.cinnamomi)、烟草疫霉(P.nicotianae)、棕榈疫霉(P.palmivora)及辣椒疫霉(P.capsici)等6种疫霉81株菌菌体可溶性蛋白质和酯酶进行了聚丙烯酰胺凝胶平板电泳研究。结果指出,种间蛋白质图谱差异明显,种内菌株间基本一致。就酯酶图谱来说,种内菌株间存在一定差异,但种间差异更为显著。因此,本试验进一步支持槐生疫霉新种的建立。同时表明,菌体可溶性蛋白质电泳图谱在疫霉种的鉴定和分类上具有重要意义,而酯酶电泳图谱在一定程度上也有助于疫霉种的研究。     

Pythium contiguanum nomen novum (syn. Pythium dreschleri Paul), its antagonism to Botrytis cinerea, ITS1 region of its nuclear ribosomal DNA, and its comparison with related species

Pythium drechsleri Paul was described as a new species from soil samples taken in a salt-marsh of Arzew, Algeria [Paul, B. (1988) Une nouvelle espèce de Pythium isolée d'une saline de l'ouest Algérien. Cryptogam. Mycol. 9, 325-333]. The name of the fungus, P. drechsleri, is a nomen invalidum, as it is a later homonym of P. drechsleri Rajgopalan and Ramakrishnan [Rajagopalan, S. and Ramakrishnan, K. (1971) Phycomycetes in agricultural soils with special reference to the Pythiaceae. Madras Univ. J. Sect. B 37,38, 100-117]. A new name, Pythium contiguanum is now being given to P. drechsleri Paul. This species is characterised by its contiguous inflated type of sporangia, smooth-walled oogonia and mostly monoclinous antheridia. Although the fungus is not known to be a pathogen, it has a very well developed appressorial system comprised of both sickle shaped and coiled appressoria. Morphological features, its antagonism towards the grape-vine pathogen Botrytis cinerea, together with the sequences of the ITS1 region of its nuclear ribosomal DNA and its comparison with related species are given here.     

Serological characterization of a ribonuclease from Hordeum vulgare

Specific rabbit antisera against purified Hordeum vulgare seedling RNase I from two winter barley cultivars each formed a single precipitin band when reacted with the homologous crude tissue extract. RNase antigen from either cultivar was equally reactive with both antisera when evaluated by immunodiffusion and immunoelectrophoresis. A small but consistent difference in anti-RNase specificity between cultivars was shown by passive hemagglutination inhibition, suggesting that molecular differences may exist between the two RNase antigens. Immunodiffusion and rocket immunoelectrophoresis were used to qualitatively test the cross-reactivity of protein preparations from various members of the genus Hordeum and species from other related grass genera. Neither antiserum showed cross-reactivity with soluble protein preparation from species outside the genus Hordeum. A few species within the genus Hordeum were cross-reactive. A modification of rocket immunoelectrophoresis was developed to determine the amount of RNase in unpurified tissue extracts. The technique involved a template-reservoir which allowed detection of 250 ng RNase in tissue extract volumes of 50 μl. The amount of RNase in unpurified protein extracts from the two cultivars of barley was similar.     

Isolation and characterization of bacterial flagellar hook proteins from salmonellae and Escherichia coli.

Flagellar hook proteins from Salmonella and Escherichia coli were dissociated in acid and purified by diethylamino-ethyl-cellulose column chromatography. These two proteins had the same electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. However, analytical electrofocusing patterns showed that these proteins had different isoelectric points (4.7 for Salmonella typhimurium and 4.4 for E. coli). Immunodiffusion and immuno-electron microscopy carried out with antisera prepared against purified hook proteins from S. typhimurium and E. coli showed that these antisera reacted with both hooks. Affinity chromatography allowed separation of antibodies specific for hook proteins from each bacterial species. These results indicate that the hook proteins share common antigenic determinants as well as specific antigens, although the specificity is not quantitatively resolved. From comparisons of the amino acid composition of the hook proteins and flagellins, it was concluded that the differences between flagellins from S. typhimurium and E. coli were larger than those between hook proteins from these species.     

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。     

Differential uptake and metabolism of sitosterol and cholesterol by Achlya, Pythium, and Phytophthora species.

The relative ability of isolates of Achlya bisexualis and A. ambisexualis and isolates of Pythium and Phytophthora to take up and metabolize sitosterol and cholesterol was studied. Species of Pythium and Phytophthora took up cholesterol and sitosterol efficiently, whereas Achlya species took up booth sterols inefficiently. Species of Pythium and Phytophthora produced a polar metabolite and esters from sitosterol as they did from cholesterol. Achlya species did not produce the polar metabolite from either sterol. In these experiments Achlya species produced esters only from cholesterol; however, their failure to produce esters from sitosterol may have been due to the higher sitosterol than cholesterol concentration.     

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.     

三种猕猴桃根腐病致病菌多重实时定量PCR检测技术的建立及应用

[背景]近年来,随着猕猴桃种植面积的不断扩大,病害的频繁发生已逐渐影响猕猴桃的产量和品质。恶疫霉(Phytophthora cactorum)、樟疫霉(P.cinnamomi)和雪松疫霉(P.lateralis)是一类可以引起猕猴桃根腐病的致病疫霉菌。[目的]建立并优化可以同时检测3种致病疫霉的多重实时定量检测技术,并调查猕猴桃主要产区的致病菌分布情况。[方法]基于Ypt1 (ras-related protein gene)基因设计恶疫霉、樟疫霉和雪松疫霉的特异性TaqMan探针和引物,建立并优化多重实时荧光定量PCR检测体系。利用近缘种检验检测体系特异性并进行灵敏度测试,应用该检测体系分析猕猴桃主要产区根际土壤中3种致病疫霉的Yt1基因含量。[结果]供测试的11个恶疫霉近缘种、11个樟疫霉近缘种、13个雪松疫霉近缘种及非目标菌种DNA样品中均无荧光信号,反应结果为阴性,而在恶疫霉、樟疫霉和雪松疫霉DNA样品中分别检测出HEX、FAM和ROX荧光信号,反应结果为阳性。三种疫霉的检测灵敏度均达到100fg。此外,通过对猕猴桃主产区陕西省周至县和眉县果园共166份土壤样品的检测发现,恶疫霉的分布最广泛且Ypt1基因含量最高,樟疫霉和雪松疫霉则相对较少。[结论]建立的猕猴桃根腐病致病疫霉多重实时定量检测体系特异性强、灵敏度高,适合于恶疫霉、樟疫霉和雪松疫霉的检测及定量分析。该技术可为猕猴桃疫霉病害的早期诊断、监测及预防提供指导。     

Hyphal branching systems in Phytophthora and other phycomycetes

The hyphal branching systems at the periphery of a fungal colony growing on cleared V-8 juice agar medium were analyzed quantitatively. A comparative study of Phytophthora cactorum, cinnamomi, P. megasperma, Pythium aphanidermatum, Saprolegnia ferax, Mucor hiemalis and Rhizopus homothallicus indicated that the characteristics of a hyphal branching system might be useful in taxa delineation.     

中国疫霉属真菌异宗配合的种的交配型

明确了异宗配合的疫霉属(Phytophthora)7个种(P.capsici,P.cinnamomi,P.drechsleri,P.melonis,P.nicotianae[=P.parasitica],P.palmivora 和 P.sinensis)47个菌株的交配型。特别有意义的是 P.drechsleri A~1和 A~2两个交配型在南京具有相同的优势,并且证实 P.cinnamomi A~1交配型的存在。     

Phytophthora irrigata, a new species isolated from irrigation reservoirs and rivers in Eastern United States of America

A new species of Phytophthora, previously referred to as the 'Dre I' taxon, is named Phytophthora irrigata. Isolates of P. irrigata morphologically and physiologically resemble Phytophthora drechsleri. They are heterothallic, produce nonpapillate sporangia, and grow well at 35 degrees C. The above two species differ significantly in uniformity of mycelium, presence of chlamydospores, DNA fingerprint, and sequences of multiple nuclear and mtDNA regions. Phytophthora irrigata produces smaller sporangia and a distinct DNA fingerprint. Sequence alignments in the ribosomal DNA internal transcribed spacer regions place Phytophthora fallax and Phytophthora captisoa as its closest relatives. The optimal temperature for culture growth is above 30 degrees C and the maximum temperature is 40 degrees C. This new species is abundant in irrigation reservoirs and natural waterways in Virginia and was also isolated in Pennsylvania.     

掘氏疫霉同宗配合、异宗配合特性的研究

本文对掘氏疫霉的同宗配合、异宗配合特性进行了研究,发现新分离的掘氏疫霉A~2交配型菌株,不经过配对培养可以产生卵孢子;菌种在15℃下保存,自身可孕性能力逐渐减退。菌株通过接种黄瓜后,异宗配合特性可以恢复,同宗配合特性不能恢复。经过营养生长,无性繁殖,菌株后代的交配型不引起改变。通过对疫霉属有关种不同交配型与掘氏疫霉各菌株不同交配型配对培养,以及菌种不同保存时间、不同培养基、疫霉属有关种的种间和种内配对,讨论了掘氏疫霉同宗配合、异宗配合特性的影响因素。     

The effect of antiserum quality on strain specificity assessment of foot and mouth disease virus by the neutralization reaction

The factors affecting the virus strain specificity of antibody to foot an mouth disease virus prepared by a variety of protocols in several species were evaluated by neutralization tests. The time at which the serum was taken, the antigen dose given, whether or not revaccination had occurred and the animal species in which the sera were prepared, did not appear to affect the strain specificity of serum prepared to inactivated antigens when measured in neutralization tests, probably because of the restricted nature of the antigenic site involved. However, variation was observed with convalescent animal sera or sera from animals which had received trypsin cleaved virus were used. For these reasons banks of reference antisera are prepared as pooled sera using one or two inoculations of inactivated antigen.     

Immunoelectrophoretic characteristics ofOphiobolus graminis Sacc. as an aid in classification and determination

Antigens from four cultures ofO. graminis were compared immunoelectro-phoretically. Each culture produced a characteristic immunogram. More common antigens were found between the two cultures isolated from wheat or the two cultures isolated from oat than between a wheat and an oat isolate. Cell-wall antigens were the best reference antigens for serologic analysis of strain relationship. O. graminis antisera were cross-reacted with antigens from a number of other species of fungi. Relatively few of these cross-reacted with antisera to cell-wall antigens whereas more cross-reacted with antisera to whole-cell antigens.Immunoelectrophoretic analysis of antigens from a range of isolates ofO. graminis indicates specific immunograms which can be determined and separated from the immunograms developed by all other fungi when tested againstO. graminis antiserum. Immunoelectrophoresis can therefore be used as an aid in determiningO. graminis.