戊唑醇对小麦赤霉菌侵染影响的细胞学研究

采用电镜技术研究了三唑类杀菌剂戊唑醇(tebuconazole)对赤霉病菌Fusarium gramineaum侵染小麦穗部过程的影响.结果表明:人工接种前2天施药,可推迟外稃内表皮、内稃及子房上分生孢子的萌发,但不能完全抑制其萌发,可引起芽管和菌丝严重畸形,不能形成侵染菌丝侵入寄主.而人工接种后2天施药,戊唑醇则严重抑制了病菌菌丝的生长,使寄主体表和寄主组织内的菌丝形态、结构发生了一系列异常变化,并最终塌陷死亡,使菌丝不能扩展到穗轴部位.接种后4天施药,病菌虽已扩展到穗轴,但戊唑醇仍对穗轴中菌丝生长具有明显的抑制作用.对赤霉毒素的免疫细胞化学标记结果表明,药剂处理与未处理的寄主和菌丝细胞中都存在有毒素,但标记密度在药剂处理的寄主和菌丝细胞中明显低于未处理对照.     

小麦穗组织中脱氧镰刀菌烯醇毒素的免疫细胞化学定位

 采用免疫细胞化学技术对禾谷镰刀菌(Fusarium graminearum)在侵染小麦穗部过程中产生的脱氧镰刀菌烯醇毒素(deoxynivalenol,DON)进行了定位分析。在接种后24h,当菌丝在外稃、内稃的内侧表面扩展而尚未侵入寄主细胞前,病菌已分泌DON,并且DON已扩散到寄主组织内。在菌丝细胞内,DON主要被定位于细胞质、线粒体及细胞壁上;在寄主细胞中DON主要分布于细胞壁、叶绿体、细胞质和内质网上。在侵染初期(接种后2 d),菌丝仅能在寄主细胞间隙扩展,随寄主组织中DON浓度的升高,寄主细胞相应发生了一系列病理变化。随寄主细胞坏死(接种后3~4d),病菌进入坏死的寄主细胞。上述结果表明,DON在禾谷镰刀菌的侵染、致病和定殖过程中起着重要的作用。毒素标记结果表明病菌产生的毒素可通过穗轴微管束组织从侵染部位向上、向下转输,毒素向上的转输量明显高于向下转输     

小菜蛾感染玫烟色棒束孢后的病征及组织病理变化

为了明确玫烟色棒束孢对小菜蛾的致病作用,分别以1×10⁵孢子/mL和1×10⁸孢子/mL浓度的玫烟色棒束孢菌株EBCL03011孢子悬浮液感染小菜蛾幼虫,观察了感病幼虫体表和体内器官组织的病变情况。结果显示,感病虫体颜色由局部浅褐色逐渐变为大面积深褐色,病虫出现缩短、强直等外部形态的变化。组织切片观察显示,接种后4 h,附着于体壁的分生孢子开始萌发入侵;16 h后,小菜蛾内表皮被分解,菌丝段进入血腔。与此同时,被幼虫取食进入消化道的分生孢子也增殖产生菌丝段,以菌丝段突破肠壁细胞,向附近的脂肪体入侵;随着玫烟色棒束孢继续在虫体内增殖,24 h后各组织器官遭到不同程度破坏;48 h后,感病幼虫死亡,虫尸体内的菌丝突破体表,在虫体外形成菌丝层。表明玫烟色棒束孢入侵小菜蛾有两条途径,第1条为体表途径,第2条为消化道途径。     

多堆柄锈菌侵染玉米的细胞学及超微结构特征

为明确玉米对多堆柄锈菌Puccinia polysora侵染后病理反应的细胞学特征,利用扫描和透射电镜技术分析了玉米自交系与多堆柄锈菌互作中二者的细胞变化过程。多堆柄锈菌对玉米的侵染主要以直接穿透叶片表皮侵入为主,少量可从气孔和细胞间隙侵入。接种后,病菌夏孢子在感病自交系叶片上快速并大量萌发,在叶表生长蔓延并侵入表皮组织细胞,7 d后形成夏孢子堆;在抗病自交系上,病菌萌发、菌丝生长均受到明显抑制,少量入侵的病菌也由于寄主细胞死亡而导致菌丝和夏孢子干瘪死亡。侵染早期在感病寄主细胞间隙出现菌丝并穿透细胞壁,在胞内产生分枝菌丝,此时寄主细胞结构正常;随着菌丝进一步扩展,叶绿体等结构发生紊乱,被侵染细胞逐渐死亡。在抗病自交系上,接菌24 h后寄主即出现过敏性坏死反应,侵入位点与周围细胞快速坏死,抑制菌丝生长蔓延;叶绿体中清晰可见深色颗粒状物质;72 h后细胞壁外侧产生大量致密的深色结晶体,应为与抗病反应相关的酚类物质。表明抗多堆柄锈菌的玉米材料可能存在2种抗病途径,即寄主与病菌互作中由分子识别引起的免疫反应和病菌侵入后的系统防卫反应。     

玫烟色拟青霉对菜青虫的侵染及致病作用

采用扫描电镜和组织切片方法研究了玫烟色拟青霉(Paecilomyces fumosoroseus)对3龄菜青虫的侵染和致病作用。结果表明:玫烟色拟青霉可通过体壁侵染虫体。3龄菜青虫在接菌8h后,附着在虫体表面的孢子开始萌发,24h后菌丝即可穿透体壁进入血腔,48h后可见虫体内有部分菌丝体。侵入虫体内的菌丝对寄主组织没有选择性。菌丝首先在入侵的血腔中生长,然后侵入邻近的脂肪体和肌肉,随菌丝在虫体内的增殖,中肠、丝腺等相继被侵染。受侵的各组织器官均发生明显的病变,如体壁分离、脂肪体变形、溶解,肌纤维排列松散,中肠上皮细胞脱落并出现许多空泡等。     

RF－1小麦品系抗赤霉病机理初探

高抗赤霉病且丰产性较好的ＲＦ－１小麦品系和泗麦２号小麦比较后发现：该品种在扬花后残留的花药极少，穗形结构松散，麦穗主轴弯曲度大，穗轴表皮下机械组织排列紧密且层次多。室内离体麦穗接种的病情扩展较对照品种慢。     

小麦品种对赤霉病的感病和抗病因素研究

 本文是报道小麦品种感染赤霉病的程度差异及其有关因素。
温室试验,在麦穗终花期分别对残留于颖外的花药;残留于颖内的花药;无残留花药的子房上,进行接种。三种处理的赤霉病发生量之比,浙麦1号(感病)为15:58:31,苏麦3号(抗病)为4:18:13。结果表明:品种间差异显著;两品种均以外露花药上接种的处理发病最轻,颖内接种.有残留花药发病加重,尤以浙麦1号为明显;两品种始病后的病情扩展速度有异,苏麦3号表现扩展慢、穗轴不受侵害。因此,一个品种的穗部全部颖花中残留花药比率低,是减缓初侵染病程的性状;小穗和穗轴组织减缓病情发展,是抗扩展性状。具备两者,则相对抗性较为稳定。
室内外对普通小麦品种不同成熟阶段的接种试验表明,抗病品种具有感病期短、乳熟期的抗性明显增强和接种后发病盛期较晚等特点。     

禾谷镰刀菌侵染引致小麦穗组织细胞壁成分变化的细胞化学研究

 本文采用细胞化学方法, 对健康和禾谷镰刀菌(Fusarium graminearum)侵染的小麦穗组织中细胞壁主要成分进行了比较分析。电镜观察发现, 被侵穗部组织细胞壁中的主要成分如纤维素、木聚糖和果胶质的标记密度下降, 显著低于未接种的健康对照组织。结果表明病菌侵染和扩展过程中分泌产生了纤维素酶、木聚糖酶和果胶酶等细胞壁降解酶类, 造成寄主细胞壁成分的分解及细胞壁松弛, 从而有利于病菌在寄主穗部组织中的侵染和扩展。     

罗汉果青枯病菌生物学特性初步研究

对罗汉果青枯病菌的生长温度、pH、碳氮营养等生物学特性进行了测定。试验结果表明,罗汉果青枯病菌Lu L3菌株能在pH5.0～8.5和12～40 ℃的范围内生长,最适生长的pH范围为6.0～7.5,最适生长的温度范围为28～35 ℃。测试的6株病原菌株,其生长均能利用蔗糖、葡萄糖、乳糖、山梨醇、麦芽糖、甜醇和甘露醇作为碳源,利用蛋白胨、牛肉浸膏、酵母浸膏、硝酸铵、硝酸钾和脲作为氮源。气温、土壤种类和罗汉果的生育期对病原菌入侵寄主有影响。在20～35 ℃范围内,气温升高利于病原菌的入侵,病害发生严重;病原菌在黏土中入侵寄主容易,在壤土中入侵寄主困难;罗汉果植株越小越利于病原菌的入侵,花蕾期后,未观察到植株被病原菌侵染。接种病原菌Lu L3菌株后,12 h时植株的根表分离到接种的病原菌株;24 h时根内分离到接种的病原菌株;36 h时茎内分离到接种的病原菌株。     

玉米感染肿囊腐霉后寄主-病原物互作的超微结构研究

 本文利用透射电镜,首次对玉米不同抗性寄主与肿囊腐霉(Pythium inflatum)相互作用中的寄主反应及菌丝在寄主内的发展进行了系统研究。结果表明:玉米苗期根部接种后,孢子迅速萌发成菌丝在根表蔓延,随即穿透根表皮,进入表皮细胞、皮层甚至感病寄主的维管束组织。与此同时,寄主相应的反应迅速,寄主反应采取如下方式:细胞壁的沉积物及乳突在真菌的入侵处形成,各种无定形物质或纤丝构成的质网包围入侵菌丝。沉积在寄主细胞壁上并在受侵染细胞内聚集的化合物可能是酚类物质,这些嗜锇酸的电子致密物是用来机械地加强细胞壁的强度及产生抗真菌的环境。寄主反应的早晚及程度的强弱决定着菌丝在寄主体内发展繁殖的程度及寄主抗性的强弱。抗病玉米自交系的反应程度及速度显著强于感病玉米。     

Infection of wheat spikes by <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> and alterations of cell wall components in the infected tissue

The infection process of Fusarium avenaceum on wheat spikes and the alteration of cell wall components in the infected host tissue were examined by means of electron microscopy and cytochemical labelling techniques following spray inoculation at growth stage (GS) 65 (mid-flowering). Macroconidia of the pathogen germinated with one to several germ-tubes 6–12 h after inoculation (hai) on host surfaces. The germ-tubes did not penetrate host tissues immediately, but extended and branched on the host surfaces. Hyphal growth on abaxial surfaces of the glume, lemma and palea was scanty 3–4 days after inoculation (dai) and no direct penetration of the outer surfaces of the spikelet was observed. Dense mycelial networks formed on the inner surfaces of the glume, lemma, palea and ovary 36–48 hai. Penetration of the host tissue occurred 36 hai by infection hyphae only on the adaxial surfaces of the glume, lemma, palea and upper part of ovary. The fungus penetrated the cuticle and hyphae extended subcuticularly or between the epidermal wall layers. The subcuticular growth phase was followed by penetration of the epidermal wall, and hyphae spread rapidly inter- and intracellularly in the glume, lemma, palea and ovary. During this necrotrophic colonization phase of the wheat spike, a series of alterations occurred in the host tissues, such as degeneration of cytoplasm and cell organelles, collapse of host cells and disintegration of host cell walls. Immunogold labelling techniques showed that cell walls of spike tissues contained reduced amounts of cellulose, xylan and pectin near intercellular hyphae or infection pegs compared to walls of healthy host tissues. These studies suggest that cell wall degrading enzymes produced by F. avenaceum facilitated rapid colonization of wheat spikes. The different penetration properties of abaxial and adaxial surfaces of the spikelet tissues as well as the two distinct colonization strategies of host tissues by F. avenaceum are discussed. The penetration and colonization behaviour of F. avenaceum in wheat spikelets resembled that of F. culmorum and F. graminearum, although mycotoxins produced by F. avenaceum differed from those of the latter two Fusarium species.     

Studies on the Infection Process of Fusarium Culmorum in Wheat Spikes: Degradation of Host Cell Wall Components and Localization of Trichothecene Toxins in Infected Tissue

After single spikelet inoculation, the infection process of Fusarium culmorum and spread of fungal hyphae in the spike tissues were studied by scanning and transmission electron microscopy. While hyphal growth on outer surfaces of the spike was scanty and no successful penetration was observed, the fungus developed a dense mycelium on the inner surfaces and effectively invaded the lemma, glume, palea and ovary by penetration pegs. During the inter- and intracellular spreading of the fungus, marked alterations in the host tissues were observed, including degeneration of cytoplasm, cell organelles, and depositions of electron dense material between cell wall and plasmalemma. Ultrastructural studies revealed that host cell walls in proximity of the penetration peg and in contact with hyphae were less dense or transparent which suggested that cell wall degrading enzymes were involved in colonisation of host tissues by fungal hyphae. Enzyme- and immunogold-labelling investigations confirmed involvement of extracellular enzymes, that is cellulases, xylanases and pectinases, in degradation of cell wall components. Localization studies of trichothecenes indicated that toxins could be detected in host tissues at an early stage of infection.     

Importance of Cell Wall Degrading Enzymes Produced by Fusarium graminearum during Infection of Wheat Heads

Cytological studies were carried out to elucidate the importance of cell wall degrading enzymes (CWDE) during infection of wheat spikes by Fusarium graminearum. Scanning electron micrographs revealed that at 6–24 hours after inoculation (hai) of single spikelets with macroconidia of F. graminearum, the fungus germinated by forming several germ tubes and developed a dense hyphal network in the cavity of the spikelet. At 24–36hai, the fungus formed infection hyphae which invaded the ovary and inner surface of the lemma and palea. Transmission electron microscopical studies revealed that the fungus extended inter- and intracellularly in the ovary, lemma and rachis and caused considerable damage and alterations to the host cell walls. In different tissues of healthy and F. graminearum-infected wheat spikes the cell wall components cellulose, xylan and pectin were localized by means of enzyme-gold and immuno-gold labelling techniques. Localization of cellulose, xylan and pectin showed that host cell walls which were in direct contact with the pathogen surface had reduced gold labelling compared to considerable higher labelling densities of walls distant from the pathogen–host interface or in non-colonized tissues. The reduced gold labelling densities in the infected host cell walls indicate that these polysaccharide degrading enzymes might be important pathogenicity factors of F. graminearum during infection of wheat spikes. The results revealed that, infection and colonization of wheat spikes by F. graminearum and reactions of infected host tissue were similar to those reported for F. culmorum.     

Immunocytochemical localization of β-1,3-glucanase and chitinase in Fusarium culmorum -infected wheat spikes

Two antisera raised against acidic β-1,3-glucanase and acidic chitinase from tobacco were used to investigate the subcellular localization of the two enzymes in Fusarium culmorum -infected wheat spike by means of the immunogold labelling technique. The studies demonstrated that the distribution of β-1, 3-glucanase and chitinase were very similar in the uninoculated healthy and infected wheat spikes. The enzymes were localized mainly in the cell walls of different tissues including the lemma, ovary and rachis of the wheat spike, while the cytoplasm and organelles of cells in these tissues showed almost no labelling. However, the accumulation of β-1,3-glucanase and chitinase in the infected wheat spikes differed distinctly between resistant and susceptible wheat cultivars. The labelling densities for the two enzymes in the infected lemma, ovary and rachis of the susceptible cultivar Agent increased only slightly as compared to the corresponding uninoculated healthy tissues, whereas higher labelling densities of β-1,3-glucanase and chitinase were found in the infected tissues of wheat spikes from the resistant cultivar Arina compared to the corresponding uninoculated healthy tissues. Furthermore, the labelling of β-1,3-glucanase and chitinase also occurred over the cell walls of the hyphae in the infected wheat spike, but not over the hyphal cytoplasm. In addition, labelling for the two enzymes was often detected over the cell wall appositions and the electron-dense material located between the host cell and the hyphal cell in the infected tissues of the resistant wheat cultivar. The findings reported in the present study indicate that β-1,3-glucanase and chitinase accumulation in the F. culmorum -infected wheat spike may be involved in resistance to pathogen spread in the host tissue.     

Distribution of Mycelial Colonies and Lesions in Field-Grown Barley Inoculated with Fusarium graminearum

ABSTRACT External surfaces of barley florets have thick-walled epidermal cells resistant to direct penetration by the head blight pathogen, Fusarium graminearum. Surfaces within the floral cavity have thin-walled, susceptible cells. How the fungus gains access to the floral cavity, causing head blight, has not been determined. To investigate pathways of entry, field-grown plants were sprayed with macroconidial inoculum after heads emerged from the flag leaf sheath and then were mist irrigated daily in the morning and evening. On selected days, 1 to 8 days after inoculation (DAI), 80 to 190 florets per day were harvested, dissected, and examined for presence and location of mycelial colonies. At 1 to 12 DAI, 57 to 100 florets likewise were examined for lesions. Patterns of colonization indicated that the fungus entered florets principally through crevices between the overlapping lemma and palea or through the apical floret mouth. The crevices were open for entry until approximately 8 days after heads emerged. Most florets had mycelial colonies on the external surface in a sheltered pocket near the base of the ventral furrow of the palea. Mycelia spread laterally from the furrow to the crevice between lemma and palea. Anther colonization had only a minor role in invasion of florets. Hyphal penetration of stomates was not seen. Lesions usually developed first within 3 mm of the floret apex or 3 mm of the floret base. Within florets, lesions often were contiguous between lemma and palea, palea and caryopsis, or in all three floret parts. However, lesions in the caryopsis developed later and were fewer in number than in the lemma and palea and always were associated with lesions in the palea. The results show the importance of initial mycelial colonization of floret outer surfaces, pathways of entry via lemma or palea crevices or floret mouth, and spread of lesions within the floret at interfaces between lemma, palea, and caryopsis.     

Rice false smut pathogen, Ustilaginoidea virens, invades through small gap at the apex of a rice spikelet before heading

Ustilaginoidea virens, the false smut pathogen of rice, produces false smut balls on spikelets after heading. To clarify how the fungus invades spikelets during the booting stage, we developed a fungal strain that expresses a green fluorescent protein gene and injected conidia from this strain into rice sheaths. Observations at 48?h post-inoculation showed many conidia were present on spikelet surfaces, and the conidia had germinated and the hyphae have gradually grown by 120?h post-inoculation. By 144?h, hyphae had invaded spikelets through their apices, via the small gap between the lemma and palea and had already reached all floral organs.     

Subcuticular-Intracellular Hemibiotrophic and Intercellular Necrotrophic Development of Colletotrichum acutatum on Almond

ABSTRACT The early infection and colonization processes of Colletotrichum acutatum on leaves and petals of two almond cultivars with different susceptibility to anthracnose (i.e., cvs. Carmel and Nonpareil) were examined using digital image analysis of light micrographs and histological techniques. Inoculated tissue surfaces were evaluated at selected times after inoculation and incubation at 20 degrees C. Depth maps and line profiles of the digital image analysis allowed rapid depth quantification of fungal colonization in numerous tissue samples. The results showed that the early development of C. acutatum on petals was different from that on leaf tissue. On petals, conidia germinated more rapidly, germ tubes were longer, and fewer appressoria developed than on leaves. On both tissues, penetration by the pathogen occurred from appressoria and host colonization was first subcuticular and then intracellular. On petals, colonizing hyphae were first observed 24 h after inoculation and incubation at 20 degrees C, whereas on leaves they were seen 48 to 72 h after inoculation. Intercellular hyphae were formed before host cells became necrotic and macroscopic lesions developed on petals >/=48 h and on leaves >/=96 h after inoculation. Histological studies complemented data obtained by digital image analysis and showed that the fungus produced infection vesicles and broad hyphae below the cuticle and in epidermal cells. In both tissues, during the first 24 to 48 h after penetration fungal colonization was biotrophic based on the presence of healthy host cells adjacent to fungal hyphae. Later, during intercellular growth, the host-pathogen interaction became necrotrophic with collapsed host cells. Quantitative differences in appressorium formation and host colonization were found between the two almond cultivars studied. Thus, on the less susceptible cv. Nonpareil fewer appressoria developed and host colonization was reduced compared with that on cv. Carmel.     

Use of Fusarium graminearum transformed with gfp to follow infection patterns in barley and Arabidopsis

The fungal pathogen Fusarium graminearum attacks the seed spikes of barley and wheat, causing sterility, reduced seed weight and accumulation of mycotoxins. To explore infection patterns in barley and in the Arabidopsis model system, the green fluorescent protein gene (gfp) was used to transform F. graminearum. Inoculation of intact barley spikes resulted in rapid colonization of the brush hairs (ovary epithelial hairs) at the extruded seed tip within 7 h. Colonization followed a pattern of rapid basipetal growth along the pericarp epithelium (interior to the lemma and palea), accompanied by slower growth inward through the pericarp and testa. However, at 16 days after infection the aleurone and starchy endosperm remained uninfected, despite heavy colonization of the pericarp. Colonization of the outer lemma also occurred but was much slower. No increase in amylase enzyme activities was found, discounting the possibility that F. graminearum utilizes gibberellin-induced host enzymes to tap the endosperm for nutrients. The transformed Fusarium strain readily infected Arabidopsis thaliana leaves and produced copious spores within distant leaves. Results show the utility of gfp in tracing the growth of this pathogen, without misinterpretation due to contaminating fungi, and for resistance studies utilizing the Arabidopsis model system.