Factors affecting in vitro maturation of isolated maize microspores

Summary An in vitro method to simulate pollen development was developed in maize (Zea mays L.). Microspores at the late uninucleate to early binucleate stage were isolated and cultured under various conditions. Cell viability, starch content and the formation of the three nuclei as found in normal mature pollen were monitored during the course of the culture. Media composition was modified in order to promote starch accumulation and frequency of mitosis, while maintaining the viability of the microspores. Under the best conditions, up to 12% of the microspores matured in vitro into trinucleate, starch-filled viable pollen grains which were unable to germinate or produce seeds. At different stages during in vitro maturation, proteins patterns were analyzed and compared with their in vivo equivalent and the patterns were only partially similar.     

梨果黑斑病菌AaCaMK基因克隆、生物信息学分析及其在侵染结构分化中的表达分析

[背景] 钙/钙调素依赖型蛋白激酶（Calcium/Calmodulin-Dependent Protein Kinase,CaMK）是真核生物细胞钙信号途径中钙调素下游的一类重要靶蛋白,对病原物生长、胁迫响应及致病性等具有重要的调控作用。[目的] 对梨果黑斑病菌互隔交链孢（Alternaria alternata）AaCaMK基因进行克隆、生物信息学分析,并对其在侵染结构分化过程中的基因表达情况进行分析,为进一步研究梨果黑斑病菌钙离子信号途径中AaCaMK对A.alternata侵染结构分化调控的分子机制提供一定的理论依据。[方法] 采用同源克隆法从A. alternata JT-03中克隆得到3个AaCaMK基因;通过TMHMM、ProtScale、SOPMA等软件对AaCaMK基因进行生物信息学分析;利用实时荧光定量PCR （RT-qPCR）技术分析AaCaMK在梨果黑斑病菌侵染结构分化过程中的表达情况。[结果] 克隆得到片段分别为1 212、1 200、2 349 bp的AaCaMK1、AaCaMK2和AaCaMK3基因;生物信息学分析表明,AaCaMK1、AaCaMK2和AaCaMK3均含有典型的蛋白激酶超家族催化结构域（PKC_Like Superfamily）,并且AaCaMK1和AaCaMK2共同含有CaMK类丝/苏氨酸蛋白激酶催化结构域（STKc_CaMK）,AaCaMK3含有LKB1/CaMKK类丝/苏氨酸蛋白激酶催化结构域（STKc_LKB1_CaMKK）;同源性分析表明,AaCaMK1、AaCaMK2和AaCaMK3分别与玉米大斑病菌CAK1、CAK2和CAK3的相似性高达94.32%、97.49%和86.57%;RT-qPCR分析表明,AaCaMK1、AaCaMK2和AaCaMK3在疏水及果蜡诱导A. alternata侵染结构分化过程中均显著上调表达（P<0.05）,而且果蜡诱导作用更显著。其中AaCaMK1和AaCaMK2在附着胞形成时期（6 h）表达量为对照的1.51倍和3.05倍,而AaCaMK3在侵染菌丝形成阶段（8 h）表达量最高,为对照的2.86倍,并且在果蜡诱导下,这3个基因在芽管伸长阶段（4 h）的上调表达量显著高于疏水界面。[结论] 钙信号中AaCaMK基因在疏水及果蜡诱导A.alternata侵染结构分化过程中发挥重要的调控作用。     

Isolation and characterization of a polygalacturonase gene highly expressed in Brassica napus pollen

A cDNA clone, Sta 44-4, corresponding to a mRNA highly expressed in Brassica napus cv. Westar stamens, was isolated by differential screening and characterized. Northern blot and in situ analyses demonstrated that Sta 44-4 is synthesized in pollen beginning at the late uninucleate stage and reaches a maximum in trinucleate microspores. Sta 44-4 displayed significant sequence similarity to known pollen polygalacturonase genes. The B. napus pollen polygalacturonase gene was shown to be part of a small gene family and to display some polymorphism among different cultivars.     

Effects of caffeine,cyclic 3′, 5′ adenosine monophosphate and cyclic 3′, 5′ guanosine monophosphate in the development of the mycelial form of Sporothrix schenckii

The kinetics of the development of the mycelial form of Sporothrix schenckii from yeast cells and conidia in a minimal basal medium with glucose at pH 4.0 and 25 °C were established. Germ tube formation was used as the index of germination for both yeast cells and conidia. Yeast cells were first observed to develop germ tubes after 3 h of incubation, reaching 92±5%, after 12 h of incubation. Germ tubes were first detected in conidia after 9 h of incubation, and 12 h after inoculation 92±6% of the conidia had germ tubes. After 24 h of incubation, fully developed, sporulating mycelia were observed from both yeast cells and conidia. A delay in germ tube formation from yeast cells was observed when But₂cAMP(10 mM) and But₂cGMP (10 mM) were added to the medium. Also the addition of caffeine, a cyclic nucleotide phosphodiesterase inhibitor, inhibited the yeast to mycelial transition. Conidial germination into the mycelial form was also inhibited when cAMP, But₂cAMP and caffeine were added to the medium. These results suggest the possible involvement of cyclic nucleotides in the control of dimorphism in S. schenckii.     

Effects of temperature on germination and hyphal growth from ascospores of A-group and B-group Leptosphaeria maculans (phoma stem canker of oilseed rape)

Ascospores of both A‐group and B‐group Leptosphaeria maculans germinated at temperatures from 5–20°C on distilled water agar or detached oilseed rape leaves. After 2 h of incubation on water agar, some A‐group ascospores had germinated at 10–20°C and some B‐group ascospores had germinated at 5–20°C. The percentages of both A‐group and B‐group ascospores that had germinated after 24 h of incubation increased with increasing temperature from 5–20°C. The observed time (Vo₅₀) which elapsed from inoculation until 50% of the spores had germinated was shorter for B‐group than for A‐group ascospores. Germ tube length increased with increasing temperature from 5–20°C for both ascospore groups. Germ tubes from B‐group ascospores were longer than germ tubes from A‐group ascospores at all temperatures tested, but the mean diameter of germ tubes from A‐group ascospores (1.8 μm) was greater than that of those from B‐group ascospores (1.2μm) at 15°C and 20°C. The average number of germ tubes produced from A‐group ascospores (3.8) was greater than that from B‐group ascospores (3.1) after 24 h of incubation at 20°C, on both water agar and leaf surfaces. Germ tubes originated predominantly from interstitial cells or terminal cells of A‐group or B‐group ascospores, respectively, on both water agar and leaf surfaces. Hyphae from A‐group ascospores grew tortuously with extensive branching, whilst those from B‐group ascospores were predominantly long and straight with little branching, whether the ascospores were produced from oilseed rape debris or from crosses between single ascospore isolates, and whether ascospores were germinating on water agar or leaf surfaces.     

Detection of protein and carbohydrate in the extracellular matrix released byCochliobolus heterostrophus during germination

The extracellular matrix (ECM) ofCochliobolus heterostrophus (anamorph:Bipolaris maydis) was made visible by gold/silver and FITC-lectin staining at different stages of germ tube development. A proteinaceous material was released from conidia as germ tubes began to emerge and continued to be released from the germ tube tip throughout elongation. A material that did not stain for protein was observed to surround germ tubes upon their elongation. At later stages of maturation, germ tubes were surrounded by a sheath of proteinaceous material. After 15 h of incubation, staining with the FITC-labeled Concanavalin A revealed that a carbohydrate material surrounded and extended between hyphae. The ECM extract was separated into two fractions which were shown by SDS-PAGE and HPLC analyses to consist of proteins and carbohydrates. The results demonstrate that the composition and physical structure of the ECM change over time. Thus, the ECM is not a static material. Rather, the components of the ECM appear to be laid down at different stages of fungal morphogenesis, possibly related to germ tube emergence, elongation, and maturation.     

Role of Pear Fruit Cuticular Wax and Surface Hydrophobicity in Regulating the Prepenetration Phase of Alternaria alternata Infection

The role of cuticular wax and the surface hydrophobicity of the fruit of the ‘Zaosu’ pear (Pyrus bretschneideri Rehd) in regulating the prepenetration phase of Alternaria alternata infection were analysed in vivo and in vitro. Results showed that cuticular wax on an intact fruit surface, as well as wax extracts mounted on silanized glass slides or onion epidermis, favoured the formation of short, differentiated germ tubes and large numbers of appressoria (APP) or infected hyphae (IH). Dewaxed fruits or no wax extract mounted on in vitro surfaces, however, enhanced germ tube elongation and inhibited or delayed the formation of infection structure. High surface hydrophobicity resulting from cuticular wax also stimulated infection structure formation, as contact angle (hydrophobicity) was positively correlated with APP formation but negatively correlated with germ tube elongation. Alternaria alternata cutinase enzyme activity was also induced by cuticular wax, both in vivo and in vitro. These findings suggest that the chemical composition and hydrophobicity of pear fruit cuticular wax are essential in facilitating fungal invasion by regulating the growth and differentiation of A. alternata during the prepenetration phase.     

水稻雄性不育与花药中类脂褐素的积累

细胞质雄性不育水稻不育系珍汕９７Ａ和其保持系珍汕９７Ｂ,处于不育期的光（温）敏核不育水稻Ｗ６１５４ｓ和培矮６４ｓ的花药中类脂褐素（ＬＦＬＰ）含量随花粉发育或败育而增高．不育花药中ＬＦＬＰ的形成速率比可育花药快,三核期的珍汕９７Ａ和不育期Ｗ６１５４ｓ的花药,其ＬＦＬＰ比相应具育性花药高２４％．用抗氧化剂ＧＳＨ、ＢＨＴ和Ｎ２处理离体的单核期花药,发现ＧＳＨ可降低珍汕９７Ａ和不育期的Ｗ６１５４ｓ的ＬＦＬＰ含量．结果认为,水稻雄性不育与膜脂过氧化作用的荧光产物类脂褐素的积累有关．     

Regeneration of the cell wall in protoplasts of Candida albicans

To assess the dynamics of synthesis of the wall by regenerating Candida albicans protoplasts deposition of chitin and mannoproteins were investigated ultrastructurally using wheat germ agglutinin conjugated with either horseradish peroxidase or colloidal gold, and Concanavalin A coupled to ferritin respectively.Freshly prepared protoplasts lacked wheat germ agglutinin receptor sites but after 1–2 h of regeneration, they were detected. After 4–5 h of regeneration, the cell wall showed a discrete structure which was only labelled with wheat germ agglutinin in thin sections. At this stage of regeneration the outermost layer of the wall was labelled with clusters of Concanavalin A-ferritin particles.After 8 h regeneration, the cell wall appeared compact, and homogenously marked with wheat germ agglutinin whereas only the surface layers appeared consistently labelled with Concanavalin A-ferritin.From these observations we conclude that C. albicans protoplasts are able to regenerate in liquid medium a cell wall consisting of a network of chitin fibrils and mannoproteins at least (glucan polymers were not determined in the present cytological study). The former are the fundamental component of the inner layers at early stages of regeneration, whereas the latter molecules are predominant in the outer layers of the wall.Abbreviations WGA-HRP wheat germ agglutinin conjugated with horseradish peroxidase - WGA-Au wheat germ agglutinin conjugated with colloidal gold - Con A-ferritin Concanavalin A coupled to ferritin     

Direct microscopic studies of clamp connection formation in growing hyphae of Schizophyllum commune

Summary Quantitative measurements of apical growth, nuclear movements and pseudo-clamp connection formation were compared with photomicroscopic details of cytoplasmic events in live A-mutant hyphae of Schizophyllum commune. Nuclear behavior was described during pseudo-clamp connection formation in uninucleate, binucleate and trinucleate hyphal apices and intercalation was shown in sub-terminal regions of these hyphae. Conventional (i.e. rearward)rd) pseudo-clamp connection formation was contrasted with forward pseudo-clamp initiation. Primary branches were shown to be initiated from uninucleate, septate pseudo-clamps. The ultrastructural aspects of A-mutant septa were delineated.     

Observation of Androgenesis in Cultured Wheat Anthers at Meiosis,Tetrad, Early Uninucleate and Trinucleate Stage

The obtaining of calluses and plantlets from cultured wheat anthersat the stages from pollen mother cell to trinucleate microspore has been reported previously. Haploids as well as diploids existed among the regenerated plantlets derivedfrom anthers at these stages. Present paper reports the study on androgenesis patter-ns of cultured anthers at meiosis, tetrad, early mid- and late uninucleate and trinucleate stage. Cytological evidence of pollen-origin of calluses produced by anthers atthese stages was given. Observation showed that meiosis of wheat anthers was able tocomplete under culture conditions, resulting in releasing microspores, from which multinucleate and multicellular pollen grains formed. In meiosis anthers, abnormal cells,including syncytium and two kinds of binueleate calls were sometimes observed. Theymight be products of abnormal meiosis and abnormal development of tapetum cells. Itwas noted that failure and/or uncomplction of forming callus wall and/or pollen wallin in vitro anthers at meiosis, tetrad and early uninucleate stage occured often. Itmight lead to the low frequency of callus induction. Mature wheat anthers (trinucleate stage) contained both normal and abnormal pollen grains (pollen dimorphism); onlythe abnormal pollen grains developed into embryoids while all the normai trinucleatepollen grains degenerated rapidly. However, the date of the frequency of equal divisionof microspores suggested that abnormal pollen (N pollen, small pollen) could not be theonly source of androgenic pollens in cultured anthers at late uninucleate and other earlier stages.     

Identification of thigmoresponsive loci for cell differentiation inUromyces germlings

Summary The sites alongUromyces appendiculatus germ tubes that are responsive to topographical induction for appressorium formation were determined using glass micropipettes. The germ tubes were perturbed with the micropipettes at different sites and durations. The most responsive region of the germ tubes for appressorium formation was within 0–10 m from the cell apex where >90% of the perturbed germ tubes developed appressoria. Furthermore, only the cell surface in contact with the substratum was responsive. Appressoria could not be induced to form, under any conditions, by perturbing cell-substratum regions of the germ tubes more than 40 m from the apex. Maximum appressorium formation occurred when the perturbing micropipette was left in place for >20 min.     

Penetration of Phytophthora cinnamomi into disease tolerant and susceptible eucalypts

The mechanisms of penetration of Phytophthora cinnamomi Rands into seedling eucalypt roots were studied by light and electron microscopy. Culture grown seedlings of root-rot tolerant Eucalyptus st johnii and root-rot susceptible Eucalyptus obliqua were inoculated with both zoospores and mycelium. Zoospores encysted on roots of both species and the germ tubes penetrated without the formation of appressoria. Swellings, previously described as appressoria, were formed when the germ tube was slow to enter the host by intracellular penetration. Vegetative hyphae penetrated both inter- and intracellularly into the zones of root elongation and differentiation, often through root hairs. Evidence of hydrolysis of the host cell-wall at the point of penetration was observed in electron micrographs. Several hours after the germ tube penetrated the epidermis, a thick plug of amorphous material formed in the germ tube slightly below the level of the outer walls of the epidermal cells, sealing off the hypha within the root. Behaviour of zoospores and germ tubes and the mechanism of penetration were similar on both hosts. Micrographs do not suggest any kind of a hypersensitive reaction by the host cells during the early stages of infection.     

Vulvovaginal candidiasis is associated with the production of germ tubes by <Emphasis Type="Italic">Candida albicans</Emphasis>

Twenty Candida albicans strains isolated from women attended at the Teaching and Research in the Laboratory of Teaching and Research in Clinical Analysis of the State University of Maringa, Paraná, Brazil, have been analyzed. Yeasts were identified by classical methods and patients subdivided into asymptomatic, vulvovaginal candidiasis(VVC) and recurrent vulvovaginal candidiasis (RVVC) groups. Yeasts were incubated in RPMI + fetal calf serum to analyze germ tubes every two hours, up to 10 h. In vitro sensitivity to fluconazole, itraconazole, ketoconazole, amphotericin B and nystatin was analyzed according to NCCLS-M27-A microdilution assay. Yeast isolated from symptomatic women produced significantly more germ tubes than asymptomatic women (P < 0.05). However, no significant difference between yeasts from VVC and RVVC occurred (P > 0.05). Variation between MIC₅₀ and MIC₉₀ of tested antifungal agents was slight among isolated yeasts, while no resistant yeasts were detected. Nevertheless, VVC yeasts were more DDS (reduced dose-dependent susceptibility) for nystatin and RVVC were more DDS for ketoconazole. Results suggest that colonization by yeast in the vagina and lack of symptoms may be partially explained by the yeast’s sparse capacity to form germ tubes, On the other hand, RVVC was not associated with antimicrobial resistance. DDS high frequency for nystatin and ketoconazole indicates that identification, and susceptibility of antifungals tests are important to management of VVC.     

Karyology and hyphal characters as taxonomic criteria in Ascomycetous black yeasts and related fungi

Mycelial development of seventy-three strains of black yeasts and related fungi were studied, and numbers of nuclei per hyphal cell were counted. Two main patterns were apparent in expanding hyphae, viz. (1) uninucleate expanding hyphal cells, septum formation strictly following mitosis, and (2) multinucleate, branched, aseptate hyphal tips, septa being formed in a later stage, leading to oligo- or uninucleate mature cells. Characteristic genera in the two groups areExophiala andAureobasidium, respectively. InZasmidium and in someRamichloridium species all mycelial cells are oligonucleate. The character is indicative for relationships at the family level in black yeasts.     

Preformed messengers inMicrosporum canis macroconidia

Macroconidia ofMicrosporum canis, when placed in a nutrient medium produce germ tubes within 4–6 h. Precursor incorporation studies showed that protein synthesis occurred prior to RNA synthesis. Sucrose density gradient analysis of wet and dry spore extracts revealed the presence of 16 % and 11 % polysomes respectively. The polysomal content increased to about 50% within 15 min of germination. Synthesis of RNA occurred only after 2 h of germination. Pool equilibration of the radioactive precursors was not limiting to these measurements. Polyadenylated RNA was isolated from macroconidia and was found to comprise 2–2.5 % of the total RNA. The poly(A)⁺ RNAs were heterodisperse and translatable in a wheat germ cell free translating system. It was concluded that macroconidia ofMicrosporum canis contain pre-formed mRNA which is translated early in germination     

Development ofPuccinia striiformis West. on nutrient agar

Uredospores of a barley isolate ofPuccinia striiformis West. were germinated on glucose peptone agar and the development of colonies was observed with a microscope. A small number (0.1–1%) of germ tubes did not abort during the first 20 h of incubation and either differentiated an infection structure or continued slowly to elongate for several days. In 4 out of 9 experiments some of these sporelings produced colonies that reached maximum size (0.15–0.20 mm) after 3 weeks at 15° C.     

Salicylic acid induced systemic resistant on peanut against Alternaria alternata

Abstract

The effect of Salicylic Acid (SA) in inducing resistance in groundnut plants against Alternaria alternata was investigated. Foliar application of SA at the concentration of 1 mM significantly reduced the leaf blight disease intensity and increased the pod yield under glasshouse conditions. Changes in the activities of phenylalanine ammonium lyase, chitinase β-1,3 glucanase and in phenolic content on groundnut after application of SA and inoculation with A. alternate were studied. In SA-treated leaves (plants) an increase in phenolic content was observed five days after challenge inoculation with A. alternata in groundnut plants pretreated with SA. There was a marked increase in chitinase and pathogen inoculation in SA-treated leaves. In chitinase, β-1,3 glucanase activities were observed in response to plants with an increase in SA treated leaves. Foliar applications of SA-induced in peroxidase and polyphenol oxidase activities were observed upon challenge inoculation with pathogen.     

<Emphasis Type="Italic">In vitro</Emphasis> biocontrol analysis of <Emphasis Type="Italic">Alternaria alternata</Emphasis> (Fr.) Keissler under different environmental conditions

The species Trichoderma harzianum was analyzed as possible biocontrol agent of Alternaria alternata under different environmental conditions (water activity and temperature). The strains were analyzed macroscopically to obtain the Index of Dominance. The analysis was completed using two microscopic techniques. T. harzianum showed dominance on contact over A. alternata at all testing temperatures and water activities tested except at 0.95 a _w and 15 °C, at which T. harzianum inhibited A. alternata at a distance. Biocontrol was governed by different mechanisms such as competition for space and nutrients, mycoparasitism, and possible antibiosis. Temperature and water activity significantly influenced fungal growth rate.     

异叶苦竹花粉管生长及双受精过程

以异叶苦竹为材料,采用扫描电镜、荧光显微镜技术及传统的石蜡制片技术,解剖观察其花粉管生长途径及双受精过程。结果表明:(1)授粉后,花粉在柱头上吸水膨胀,约30 min即可萌发。(2)授粉1～2 h后花粉管可达到花粉长度的5～10倍,花粉管在柱头分支中进一步伸长,并开始伸入花柱中生长。(3)授粉后5 h,大量花粉管沿引导组织进入花柱基部与子房顶部之间的子房壁,有少量花粉管在子房壁与外珠被之间的缝隙中生长。(4)授粉后8 h,少量花粉管到达珠孔端。(5)授粉后15～18 h,精核与极核融合,形成初生胚乳核;精、卵核融合,形成合子。(6)授粉后20～30 h,仍可在花柱中见到大量呈束状的花粉管。(7)授粉后48 h,子房内的大部分花粉管出现解体,大多数花粉死亡。研究认为,精细胞到达胚珠的时间为8 h。