禾谷多粘菌游动孢子超微结构观察

本文描述了寄生在大麦根部的禾谷多粘菌Polymyxa graminis Led.的次生游动孢子的超微结构,包括核、内质网、高尔基体、线粒体、脂质粒、排泄泡、小囊、具膜小囊、核糖体以及鞭毛基体(Kinetosome)和鞭毛杆等细胞器。游动孢子中未见微体。同时也在电镜下观察了游动孢子接触和穿透根细胞时所形成的管腔(Rohr)和棘杆(Stachel)以及游动孢子穿透细胞壁的详细过程。     

全白绒泡菌部分生活史图解

为深入了解全白绒泡菌生活史过程,对其孢子萌发、黏变形体和游动胞的相互转变、游动胞在5 s内的运动轨迹及原质团形成孢囊的过程进行了显微镜下观察和拍照,并对部分生活史阶段进行了时间测定,提出黏菌孢子萌发的第三种方式,提供了清晰的全白绒泡菌部分生活史的图片。     

黄柄钙皮菌的生活史

利用基物培养、燕麦-琼脂培养技术及扫描电镜技术研究了黄柄钙皮菌的个体发育过程,在燕麦琼脂培养基上完成了从孢子到孢子的生活史。结果表明,生活史包括单核的黏变形体或游动胞、多核的营养体原质团以及孢子形成阶段。孢子球形,表面具疣突。孢子萌发为裂式,释放1黏变形体。黏变形体行变形运动,在有水的条件下,可转变为游动胞并游动。可观察到1长具极性的鞭毛。合子形成原质团。成熟原质团棕色。原质团类型为显型,具有扇形网络状菌脉。琼脂培养基上获得的黄柄钙皮菌孢子与野生型相似,并具有可育性。     

马铃薯晚疫病菌单孢分离物生物学特性的初步研究*

研究了马铃薯晚疫病菌单游动孢子分离的方法,并对其单游动孢子分离物菌落生长直径、产孢量以及对甲霜灵的敏感性进行了初步研究。结果表明同一菌株的不同单游动孢子菌落生长直径和产孢量有显著差异,但不同游动孢子分离物对甲霜灵敏感性没有显著差异。     

马铃薯晚疫病菌单孢分离物生物学特性的初步研究

研究了马铃薯晚疫病菌单游动孢子分离的方法,并对其单游动孢子分离物菌落生长直径、产孢量以及对甲霜灵的敏感性进行了初步研究。结果表明同一菌株的不同单游动孢子菌落生长直径和产孢量有显著差异,但不同游动孢子分离物对甲霜灵敏感性没有显著差异。     

大麦根中禾谷多粘菌休眠孢子堆的超微结构*

通过扫描电镜和透射电镜观察了禾谷多粘菌PolymyxagraminisLed．休眠孢子堆的超微结构。休眠孢子堆仅分布于寄主根表皮细胞中。休眠孢子堆形状不一,有的呈球状,有的呈律状,少则由几十个,多则由数百个紧密排列的休眠孢子组成。休眠孢子彼此通过刺突连接,细胞壁分4层,第三层局部区域结构松散,可能与初生游动孢子萌发孔有关。成熟休眠孢子细胞质丰富,细胞质膜内侧含有大量脂质粒,细胞质中央含一个细胞核,围围分布线粒体、内质网、高尔基体、液泡等细胞器。成熟的休眠孢子在越夏前大多数已释放初生游动孢子,只剩下空壳。表面凹陷是已释放游动孢子的休眠孢子一个特征。本文还讨论了禾谷多粘菌休眠孢子在病害流行学中的作用。     

大麦根中禾谷多粘菌休眠孢子堆的超微结构

通过扫描电镜和透射电镜观察了禾谷多粘菌PolymyxagraminisLed．休眠孢子堆的超微结构。休眠孢子堆仅分布于寄主根表皮细胞中。休眠孢子堆形状不一,有的呈球状,有的呈律状,少则由几十个,多则由数百个紧密排列的休眠孢子组成。休眠孢子彼此通过刺突连接,细胞壁分4层,第三层局部区域结构松散,可能与初生游动孢子萌发孔有关。成熟休眠孢子细胞质丰富,细胞质膜内侧含有大量脂质粒,细胞质中央含一个细胞核,围围分布线粒体、内质网、高尔基体、液泡等细胞器。成熟的休眠孢子在越夏前大多数已释放初生游动孢子,只剩下空壳。表面凹陷是已释放游动孢子的休眠孢子一个特征。本文还讨论了禾谷多粘菌休眠孢子在病害流行学中的作用。     

棕囊藻属(Phaeocystis)的分类与生活史(综述)

棕囊藻属Phaeocystis（定鞭藻纲Prymnesiophyceae)的分类问题目前还有争论。其种的分类标准是以初始的群体形态、地理分布、细胞特征等以及分子生物学特征,如染色体倍性,基因组大小等为依据。基于以上各种分类特征,目前比较确定的棕囊藻属藻类有四种：一种是只观察到单细胞形态的凹孔棕囊藻（P.scrobiculata),另外三种是能够形成群体的波切棕囊藻（P.pouchetii)、球形棕囊藻（P.globosa)和南极棕囊藻（P.antarctica)。棕囊藻具有一个复杂的异形生活史,介于几种游离的单细胞（不动的细胞,具有鞭毛的动细胞,小游动孢子以及可能存在的大游动孢子）和群体之间的形态交替。但其生活史中仍有许多不确定的问题。     

蚊幼虫病原真菌贵阳腐霉的生物学研究

为了开发灭蚊真菌贵阳腐霉Pythiumguiyangense用于蚊虫防治,对贵阳腐霉生物学特性进行了研究。采用菌丝生长率、真菌产孢情况以及对致倦库蚊Culexquinquefaciatus1龄幼虫的毒力作为评价的指标。测试了7种人工培养基、7种单糖、7种氮源、真菌生长所需的pH范围和温度范围、以及4种光-暗比的光照程序对真菌的影响。结果证明该真菌生长的适合温度为5℃～35℃,最佳温度为25℃～30℃;适合pH范围是5～12;在pH9～11范围内菌丝和游动孢子生长最好;测试的人工培养基中,按照菌丝生长速度从高到低排列,依次为Czapek’SFE、PYG、KPYG2、SDAY、CMA和PDA。其中从真菌的游动孢子形成量和对蚊幼虫的毒力来看,最佳培养基为SFE;葡萄糖、果糖、乳糖、蔗糖、麦芽糖、甘露糖和可溶性淀粉都是本真菌的合适碳氢营养源;含有机氮的培养基比含无机氮的培养基好;不同的光照程序没有表现明显的影响,但是观察到紫外光对本真菌有明显的抑制作用。     

1株裂殖壶菌(Schizochytrium sp.的分离鉴定*

采用松树花粉诱集法从乐清湾红树林分离到一株纯培养物,其特点为:营养菌体为椭圆球形,单核;营养菌体的细胞壁由许多紧压在一起的致密鳞片层构成,在细胞壁不连续处可分辨鳞片;营养菌体形成外质网,它产生于外质网形成体;营养菌体以产生游动孢子行无性繁殖,游动孢子为双鞭毛;无性繁殖过程中形成四分体结构。据此鉴定为裂殖壶菌(Schizochytrium sp·)。     

A monoclonal antibody and the lectin wheat germ agglutinin induce zoospore encystment in Pythium porphyrae, a marine microbial pathogen

Pythium porphyrae (Oomycota) is a microbial pathogen which causes red rot disease in the commercially cultivated red seaweed Porphyra. This disease is initiated by the motile zoospores of the fungus, which it has been suggested to recognize and process host specific signals by membrane bound receptors. Monoclonal antibodies (MAbs) were developed against the surface components of zoospores and cysts of this fungus in order to try and identify the putative receptor molecules involved in the zoospore encystment process. Screening of MAbs by immunofluorescence assays has revealed three different patterns of surface epitope binding, while labeling of zoospore and cysts components by FITC-conjugated lectins has identified different carbohydrate moieties. Of the MAbs and lectins tested, MAb 1A3 and wheat germ agglutinin have induced zoospore encystment under in vitro conditions. MAb 1A3 identified a 109 KDa band of a glycoprotein in western blot analysis which could be a putative receptor responsible for the induction of zoospore encystment.     

THALASSOCHYTRIUM GRACILARIOPSIDIS (CHYTRIDIOMYCOTA), GEN. ET SP. NOV., ENDOSYMBIOTIC IN GRACILARIOPSIS SP. (RHODOPHYCEAE)

Thalassochytrium gracilariopsidis gen. et sp. nov. is an endosymbiotic, polycentric, zoosporic fungus that infected cultures of the red alga Gracilariopsis sp. Based on the posteriorly uniflagellate zoospore and the platelike cristae of the mitochondria, the fungus is placed in the Chytridiomycota. Ultrastructurally, the fungal zoospore is distinguished by the anterior position of the kinetosome, a unique microbody-lipid globule complex, an electron-opaque helix associated with the kinetosome and lipid globules, and a beaked nucleus. Zoospores are positively phototactic, and the unusual helix might constitute part of the photosensory apparatus. Zoospores lack certain taxonomically important structures, such as a rumposome, props, a nonflagellated kinetosome, and flagellar roots. The organism does not fit into any described genus, and the features of its zoospore differ from those of any described order. The fungal thallus is polycentric with multinucleate, septate hyphae. Haustoria form within the algal cells. The fungus does not appear to cause major harm to its host and seems to be host specific. However, during intense sporulation of the fungus, degradation of host chloroplasts was observed in medullary cells.     

德里腐霉游动孢子诱发试验的研究

本试验研究了不同培养基、菌龄大小、温度高低、诱饵种类和预处理时间长短对德里腐霉(Pythium.deliense)游动孢子产生的影响。结果显示,CMA、WA、PDA、TSA和LBA培养基均适宜于培养P.deliense并使其产生较多量的游动孢子,其中尤以CMA和WA效果为好;幼龄培养物的产孢能力比老龄的强,并以菌龄2天为最好;此菌在8℃以下和35℃以上不产孢,15—25℃为其产孢适宜温度;以马唐叶和胡萝卜为诱饵的处理,其孢子始见期和产孢量均明显比对照组早和高,马唐叶的刺激效应较胡萝卜的为强;随着预处理时间的增加,产孢量随之相应增多,但龄期重迭现象则越严重。     

Metabolic changes during development of Phytophthora palmivora examined by gas chromatography/mass spectrometry

Metabolic profiles from four stages of differentiation of the fungus Phytophthora palmivora were obtained by gas chromatography/mass spectrometry. The profiles showed the presence of sterols in the asexual reproduction stage of the organism, and confirmed their virtual absence from the mycelial stages. The zoospore stage was characterized by the presence of polyunsaturated fatty acids of C20 and C22 chain length. The transition from zoospore to cyst was also marked by the appearance of disaccharides and by a decrease in the amount of phosphate present. There were also distinctive shifts in the proportions and the total amounts of amino acids present, with gamma-aminobutyrate and alanine increasing as germination took place. These distinctive profiles identify some of the metabolic changes which accompany differentiation in this fungus.     

Characterization of Aspergillus oryzae fermentation extract effects on the rumen fungus Neocallimastix frontalis,EB 188. Part 2. Carbon source utilization and effects on zoospore production

The effect of a commercial Aspergillus oryzae fermentation extract on the utilization of carbon source and zoospore production by the rumen fungus Neocallimastix frontalis EB 188 was determined. In addition, the composition of a soluble extract prepared from the commercial product was analyzed. This extract was added to N. frontalis EB 188 cultures grown on a variety of substrates and periodically assayed for protein, enzymes, zoospore production, and carbon source utilization. The powdered product contained 93% dry matter, more than 3,000 A. oryzaespores per gram, and did not contain strong buffers or high concentrations of salt. Measurable concentrations of DNA, protein, carbohydrate and several enzymes including cellulase and amylase were also found. Soluble extract increased fungal physiology and treated cultures produced significantly higher levels of supernatant protein and enzymes including amylase, cellulase and beta-glucosidase. The fungal response depended on culture carbon source. However, culture zoospore production was increased regardless of substrate provided. Culture utilization of glucose was more rapid in treated cultures, yet high levels of the extract greatly inhibited glucose utilization.     

In vitro production of zoospores by the mosquito pathogen Lagenidium giganteum (Oomycetes: Lagenidiales) on solid media

Reliable, large-scale production of Lagenidium giganteum zoospores was obtained on solid media. The fungus was grown for 7 days in a liquid medium of wheat germ, hemp seed, yeast extract, and glucose, then placed onto hemp-seed agar. Zoosporogenesis was induced on agar by immersing the fungal cultures into water. Zoospore production began 10 hr postimmersion, peaked at 18 hr, and ceased by 36 hr. A single, 10-cm Petri dish of fungus on hemp-seed agar produced 1.7?3.8 × 10⁷ zoospores during the 26 hr of zoosporogenesis. Optimal zoospore production occurred with 4- to 7-day-old cultures; cultures older than 10 days produced few zoospores. The temperature range for zoosporogenesis was 15–35°C. The extent of zoosporogenesis was directly related to the volume of water used to induce zoospore formation and inversely proportional to agar thickness. Bioassay of zoospores against second instar Culex quinquefasciatus larvae yielded an LD₅₀ of 400 zoospores/ml.     

The Influence of Temperature on Phytophthora infestans (Mont.) de Bary

The influence of temperature on the growth rate, sporulation density and zoospore release of Phytophthora infestans, cultivated on rye agar, has been studied. Temperature significantly influenced all the features of the fungus mentioned above. The highest yield of sporangia per 1 cm² of aerial mycelium occurred at 24°C while the highest percentage of sporangia releasing zoospores was observed when the fungus was grown at 15 °C. When considering the size of the fungal colony the highest production of sporangia was obtained at 20°C. It was concluded that the temperature at which the fungus was cultured predetermined the way it germinated.     

Identification of specific cucumber necrosis virus coat protein amino acids affecting fungus transmission and zoospore attachment

Cucumber necrosis virus (CNV) is naturally transmitted in the soil by zoospores of the fungal vector Olpidium bornovanus. Successful transmission requires that virus particles attach to the surface of zoospores prior to zoospore encystment on host roots. Mechanically passaged CNV was screened for mutants deficient in fungus transmission. We found six such mutants, exhibiting transmission efficiencies ranging from approximately 14 to 76% of that of wild-type (WT) CNV. Results of in vitro virus-zoospore binding assays show that each mutant binds to zoospores less efficiently than WT CNV (21 to 68%), suggesting that defects in transmission for these mutants are at least partially due to inefficient zoospore binding. Analysis of the structure of the CNV coat protein subunit and trimer indicates that affected amino acids in all of the mutants are located in the shell or protruding domain and that five of six of them are potentially exposed on the surface of the virus particle. In addition, several of the mutated sites, along with a previously identified site in a region of subunit-subunit interaction in the coat protein shell domain (M. A. Robbins, R. D. Reade, and D. M. Rochon, Virology 234:138-146, 1997), are located on the particle quasi-threefold axis, suggesting that this region of the capsid may be important in recognition of a putative zoospore receptor. The individual sites may directly affect attachment to a receptor or could indirectly affect attachment via changes in virion conformation.     

A Cyclic GMP-Dependent K+ Channel in the Blastocladiomycete Fungus Blastocladiella emersonii

Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor (l-cis-diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K⁺ channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K⁺ selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae, possess genes encoding a similar K⁺ channel and the rhodopsin–guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K⁺ channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus.     

ITS1 Copy Number Varies among Batrachochytrium dendrobatidis Strains: Implications for qPCR Estimates of Infection Intensity from Field-Collected Amphibian Skin Swabs

Genomic studies of the amphibian-killing fungus (Batrachochytrium dendrobatidis, [Bd]) identified three highly divergent genetic lineages, only one of which has a global distribution. Bd strains within these linages show variable genomic content due to differential loss of heterozygosity and recombination. The current quantitative polymerase chain reaction (qPCR) protocol to detect the fungus from amphibian skin swabs targets the intergenic transcribed spacer 1 (ITS1) region using a TaqMan fluorescent probe specific to Bd. We investigated the consequences of genomic differences in the quantification of ITS1 from eight distinct Bd strains, including representatives from North America, South America, the Caribbean, and Australia. To test for potential differences in amplification, we compared qPCR standards made from Bd zoospore counts for each strain, and showed that they differ significantly in amplification rates. To test potential mechanisms leading to strain differences in qPCR reaction parameters (slope and y-intercept), we: a) compared standard curves from the same strains made from extracted Bd genomic DNA in equimolar solutions, b) quantified the number of ITS1 copies per zoospore using a standard curve made from PCR-amplicons of the ITS1 region, and c) cloned and sequenced PCR-amplified ITS1 regions from these same strains to verify the presence of the probe site in all haplotypes. We found high strain variability in ITS1 copy number, ranging from 10 to 144 copies per single zoospore. Our results indicate that genome size might explain strain differences in ITS1 copy number, but not ITS1 sequence variation because the probe-binding site and primers were conserved across all haplotypes. For standards constructed from uncharacterized Bd strains, we recommend the use of single ITS1 PCR-amplicons as the absolute standard in conjunction with current quantitative assays to inform on copy number variation and provide universal estimates of pathogen zoospore loads from field-caught amphibians.