The development of a rapid PCR assay for detection of Fusarium moniliforme

The fungus Fusarium moniliforme infects a wide range of crops throughout the world. In maize (Zea mays L.) it causes seedling blight and root, stalk, and ear rots. A simple procedure that can be used to detect infection by F. moliliforme from infected plant tissues has been developed. A F. moniliforme genomic library was prepared and used to identify the recombinant clones containing fungal DNA sequences not hybridizing with the DNA of the host plant, maize. Based on the nucleotide sequence information obtained from the F. moniliforme pUCF2 genomic clone, specific oligonucleotides were designed and used as primers for in vitro DNA amplification by the polymerase chain reaction. An amplification product was obtained with F. moniliforme DNA preparations whereas no amplified DNA was detected with DNAs from other fungal pathogens, including various Fusarium species, or from the host plant. This PCR analysis was successfully employed to identify F. moniliforme directly from the mycelia that develop from naturally infected maize seeds, with no need to obtain pure fungal cultures for reliable diagnosis. The protocol can be used for the diagnosis of infected plants and soils in epidemiological studies of Fusarium diseases, for seed health testing, and for evaluation of susceptibility to colonization in commercial maize hybrids.     

玉米叶片受新月弯孢菌侵染后的细胞病理学变化

 本文利用透射电子显微镜技术与细胞化学技术研究了玉米叶片受弯孢菌侵染后的超微结构和细胞壁的组成成份变化。透射电镜观察发现,病菌侵入后,菌丝先在寄主细胞间扩展,随着寄主细胞病变、坏死,菌丝可进入寄主细胞形成胞内菌丝。随病菌侵入和在寄主体内扩展,寄主细胞先后发生了一系列的超微结构变化,叶绿体、液泡等细胞器解体,出现质壁分离现象,并最终解体、坏死、变形。细胞化学标记定位发现,受侵寄主细胞壁中纤维素、木聚糖和果胶质的标记密度明显低于未接种的健康组织,表明细胞壁降解酶(如纤维素酶、木聚糖酶和果胶酶)的产生与病菌侵染和致病过程密切相关。     

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。     

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。     

Mechanisms of Induced Resistance in Barley Against Drechslera teres

ABSTRACT Quantitative and qualitative histopathological methods and molecular analyses were used to study the mechanisms by which preinoculation with either of the nonbarley pathogens, Bipolaris maydis and Septoria nodorum, inhibited growth of Drechslera teres. Collectively, our data suggest that induced resistance is the principal mechanism responsible for impeding the pathogen. The enhancement of resistance in the host was primarily manifested during penetration by D. teres, and after penetration, where growth of D. teres ceased soon after development of infection vesicles. Thus, 24 h after pretreatment with B. maydis or S. nodorum, the penetration frequency from D. teres appressoria was reduced from 42.7% in the controls to 9.5 and 14.8%, respectively. The reductions were associated with increased formation of fluorescent papillae in induced cells (early defense reaction). The postpenetrational inhibition of D. teres completely stopped fungal growth and was apparently linked to an enhancement of multicellular hypersensitive responses in inducer-treated leaves (late defense reaction). Papillae formation and multicellular hypersensitive reactions were also observed in fully susceptible, noninduced control leaves, but they were inadequate to stop fungal progress. Northern blots from leaves treated with either inducer alone support the conclusion that induced resistance is involved in suppression of D. teres by increased formation of papillae and hypersensitive reactions. Thus, the blots showed strong expression of several defense response genes that are involved in these reactions in barley attacked by Erysiphe graminis f. sp. hordei.     

Treatment with the Mycoparasite Pythium oligandrum Triggers Induction of Defense-Related Reactions in Tomato Roots When Challenged with Fusarium oxysporum f. sp. radicis-lycopersici

ABSTRACT The influence exerted by the mycoparasite Pythium oligandrum in triggering plant defense reactions was investigated using an experimental system in which tomato plants were infected with the crown and root rot pathogen Fusarium oxysporum f. sp. radicis-lycopersici. To assess the antagonistic potential of P. oligandrum against F. oxysporum f. sp. radicis-lycopersici, the interaction between the two fungi was studied by scanning and transmission electron microscopy (SEM and TEM, respectively). SEM investigations of the interaction region between the fungi demonstrated that collapse and loss of turgor of F. oxysporum f. sp. radicis-lycopersici hyphae began soon after close contact was established with P. oligandrum. Ultrastructural observations confirmed that intimate contact between hyphae of P. oligandrum and cells of the pathogen resulted in a series of disturbances, including generalized disorganization of the host cytoplasm, retraction of the plasmalemma, and, finally, complete loss of the protoplasm. Cytochemical labeling of chitin with wheat germ agglutinin (WGA)/ovomucoid-gold complex showed that, except in the area of hyphal penetration, the chitin component of the host cell walls was structurally preserved at a time when the host cytoplasm had undergone complete disorganization. Interestingly, the same antagonistic process was observed in planta. The specific labeling patterns obtained with the exoglucanase-gold and WGA-ovomucoid-gold complexes confirmed that P. oligandrum successfully penetrated invading cells of the pathogen without causing substantial cell wall alterations, shown by the intense labeling of chitin. Cytological investigations of samples from P. oligandrum-inoculated tomato roots revealed that the fungus was able to colonize root tissues without inducing extensive cell damage. However, there was a novel finding concerning the structural alteration of the invading hyphae, evidenced by the frequent occurrence of empty fungal shells in root tissues. Pythium ingress in root tissues was associated with host metabolic changes, culminating in the elaboration of structural barriers at sites of potential fungal penetration. Striking differences in the extent of F. oxysporum f. sp. radicis-lycopersici colonization were observed between P. oligandrum-inoculated and control tomato plants. In control roots, the pathogen multiplied abundantly through much of the tissues, whereas in P. oligandrum-colonized roots pathogen growth was restricted to the outermost root tissues. This restricted pattern of pathogen colonization was accompanied by deposition of newly formed barriers beyond the infection sites. These host reactions appeared to be amplified compared to those seen in nonchallenged P. oligandrum-infected plants. Most hyphae of the pathogen that penetrated the epidermis exhibited considerable changes. Wall appositions contained large amounts of callose, in addition to be infiltrated with phenolic compounds. The labeling pattern obtained with gold-complexed laccase showed that phenolics were widely distributed in Fusarium-challenged P. oligandrum-inoculated tomato roots. Such compounds accumulated in the host cell walls and intercellular spaces. The wall-bound chitin component in Fusarium hyphae colonizing P. oligandrum-inoculated roots was preserved at a time when hyphae had undergone substantial degradation. These observations provide the first convincing evidence that P. oligandrum has the potential to induce plant defense reactions in addition to acting as a mycoparasite.     

大豆疫霉菌对大豆下胚轴侵染过程的细胞学研究

 接种后1.5~24h,用光镜和电镜研究了2个大豆品种与大豆疫霉菌Ps411的亲和性和非亲和性互作。观察结果表明,大豆疫霉菌对大豆下胚轴的侵染过程可分为侵入前、侵入、皮层组织中的扩展和进入维管束组织4个连续阶段。大豆下胚轴接种后在25℃保湿培养,1.5h后游动孢子即形成休止孢并萌发产生附着孢,3h后侵入表皮细胞,6h后进入皮层组织,24h后进入维管束组织。病原菌主要以侵染菌丝直接侵入表皮,表皮细胞间隙是主要侵入部位。皮层细胞是病原菌定殖和发展的主要场所,胞间菌丝侵入皮层细胞并形成吸器。在菌丝与寄主细胞接触部位的寄主细胞壁与质膜之间常有胞壁沉积物的形成。在抗病品种上病菌的侵染事件与感病品种基本一致,但不能形成正常的吸器,胞壁沉积物明显多于感病品种,菌丝在寄主组织内的扩展明显受到抑制。利用β-1,3-葡聚糖免疫金标记单克隆抗体进行的免疫细胞化学的研究表明,胞壁沉积物内含有大量的β-1,3-葡聚糖,在大豆疫霉菌菌丝壁中也存在β-1,3-葡聚糖。以上结果表明,病原菌的侵染可诱导抗病寄主细胞内β-1,3-葡聚糖迅速的合成与积累、并形成胞壁沉积物,以抵御病菌的侵染与扩展。     

Bacterial-Mediated Induced Resistance in Cucumber: Beneficial Effect of the Endophytic Bacterium Serratia plymuthica on the Protection Against Infection by Pythium ultimum

ABSTRACT The potential of the endophytic bacterium Serratia plymuthica strain R1GC4 in stimulating defense reactions in cucumber (Cucumis sativus) seedlings inoculated with the soilborne pathogen Pythium ultimum was explored at the cellular level. Bacterial treatment prior to Pythium inoculation resulted in less seedling disease development as compared with that in nontreated control plants, in which typical root symptoms were visible by 3 days after inoculation with the pathogen. Histological investigations of root samples revealed striking differences in the extent of plant defense reactions between bacterized and nonbacterized plants. These observations were further confirmed at the ultrastructural level with the demonstration that restriction of fungal colonization to the outermost root tissues of bacterized seedlings correlated with the deposition of enlarged callose-enriched wall appositions at sites of potential pathogen penetration and the accumulation of an osmiophilic material in the colonized areas. Hyphae of the pathogen, surrounded by this electron-opaque material, exhibited considerable changes including cytoplasm disorganization and, in many cases, loss of the protoplasm. However, labeling with the beta-1,4-exoglucanase resulted in a regular labeling of Pythium cell walls, even at a time when these walls were entirely coated by the osmiophilic material. This material was also found to infiltrate into the invading hyphae to form either an internal coating of the cell wall or a network of polymorphic droplets in the area previously occupied by the cytoplasm. Cytochemical investigations revealed that callose, pectin, and cellulose appeared in the wall appositions. In addition, glucosides, lipids, and phenolics were detected in the electron-dense aggregates forming the core of most wall appositions. Finally, galactose residues were among the minor polysaccharidic compounds detected in the wall appositions. Evidence is provided in this study showing that treatment with S. plymuthica sensitizes susceptible cucumber plants to react more rapidly and more efficiently to Pythium attack through the formation of physical and chemical barriers at sites of potential fungal entry.     

Ultrastructural and immunocytochemical investigation of pathogen development and host responses in resistant and susceptible wheat spikes infected by Fusarium culmorum

Pathogen development and host responses in wheat spikes of resistant and susceptible cultivars infected by Fusarium culmorum causing Fusarium head blight (FHB), were investigated by means of electron microscopy as well as immunogold labelling techniques. The studies revealed similarities in the infection process and the initial spreading of the pathogen in wheat spikes between resistant and susceptible cultivars. However, the pathogen’s development was obviously more slow in the resistant cultivars as in comparison to a susceptible one. The structural defence reactions such as the formation of thick layered appositions and large papillae were essentially more pronounced in the infected host tissues of the resistant cultivars, than in the susceptible one. β -1,3-glucan was detected in the appositions and papillae. Furthermore, immunogold labelling of lignin demonstrated that there were no differences in the lignin contents of the wheat spikes between susceptible and resistant cultivars regarding the uninoculated healthy tissue, but densities of lignin in host cell walls of the infected wheat spikes differed distinctly between resistant and susceptible cultivars. The lignin content in the cell walls of the infected tissues of the susceptible wheat cultivar increased slightly, while the lignin accumulated intensely in the host cell walls of the infected wheat spikes of the resistant cultivars. These findings indicate that lignin accumulation in the infected wheat spikes may play an important role in resistance to the spreading of the pathogen in the host tissues. Immunogold labelling of the Fusarium toxin DON in the infected lemma showed the same labelling patterns in the host tissues of resistant and susceptible cultivars. However, there were distinct differences in the toxin concentration between the tissues of the susceptible and resistant cultivars. At the early stage of infection, the labelling densities for DON in resistant cultivars were significantly lower than those in the susceptible one. The present study indicates that the FHB resistant cultivars are able to develop active defence reactions during infection and spreading of the pathogen in the host tissues. The lower accumulation of the toxin DON in the tissues of the infected spikes of resistant cultivars which results from the host’s defence mechanisms may allow more intensive defence responses to the pathogen by the host.     

Ultrastructural and cytochemical investigations of pathogen development and host reactions in susceptible and partially-resistant carrot roots infected by Pythium violae, the major causal agent for cavity spot

Two carrot genotypes, cultivar Nanco and line 24, susceptible and partially- resistant respectively to cavity spot, were compared ultrastructurally and cytochemically 24 h, 48 h and 72 h after root inoculation with a virulent Pythium violae isolate. The extent of pathogen ingress and the response of the host differed markedly with the two genotypes. In cv Nanco, growth of fungal hyphae was predominantly intracellular and was accompanied by pronounced damage; by 48 h after inoculation, pericycle and the first cell layers of the phloem parenchyma were invaded, resulting in host wall dissolution and cytoplasm aggregation. The growth of P. violae in line 24 was limited to the pericycle, even up to 72 h after inoculation; fungal colonization was accompanied by retraction of cytoplasm and in the appearance of granular or fibrillar material in the host cell lumen. Some affected host cells were filled with structureless osmophilic material. In cultivar Nanco, invading fungal hyphae were unaffected; by contrast in line 24, the cytoplasm of invading hyphae, particularly those inside the cell host, was disorganised and structureless. Infection and host response in the two cultivars were studied with two specific labels: Aplysia gonad lectin (AGL), a polygalacturonic acid-binding lectin, and an exoglucanase complexed to colloidal gold were used to locate pectin and cellulosic -(1,4)-glucans respectively in infected tissues. The decrease of cytochemical labeling beyong fungal penetration showed clearly hydrolysis of pectin and cellulose in cell walls of the cv Nanco. By contrast, the cell wall of line 24 remained largely intact, although, unlabeled amorphous and electron-dense material was observed inside the wall. Fibrillar or electron dense material commonly observed in infected tissue of line 24 apparently did not contain pectic or cellulosic substances. Moreover, material observed in host cells or fungal hyphae was also free of labeling. The origin and the chemical composition of these compounds as well as their possible role in the defence mechanisms of carrot against P. violae are discussed.     

Host Barriers and Responses to Uncinula necator in Developing Grape Berries

ABSTRACT Grape berries are highly susceptible to powdery mildew 1 week after bloom but acquire ontogenic resistance 2 to 3 weeks later. We recently demonstrated that germinating conidia of the grape powdery mildew pathogen (Uncinula necator) cease development before penetration of the cuticle on older resistant berries. The mechanism that halts U. necator at that particular stage was not known. Several previous studies investigated potential host barriers or cell responses to powdery mildew in berries and leaves, but none included observation of the direct effect of these factors on pathogen development. We found that cuticle thickness increased with berry age, but that ingress by the pathogen halted before formation of a visible penetration pore. Cell wall thickness remained unchanged over the first 4 weeks after bloom, the time during which berries progressed from highly susceptible to nearly immune. Autofluorescent polyphenolic compounds accumulated at a higher frequency beneath appressoria on highly susceptible berries than on highly resistant berries; and oxidation of the above phenolics, indicated by cell discoloration, developed at a significantly higher frequency on susceptible berries. Beneath the first-formed appressoria of all germinated conidia, papillae occurred at a significantly higher frequency on 2- to 5-day-old berries than on 30- to 31-day-old fruit. The relatively few papillae observed on older berries were, in most cases (82.8 to 97.3%), found beneath appressoria of conidia that had failed to produce secondary hyphae. This contrasted with the more abundantly produced papillae on younger berries, where only 35.4 to 41.0% were located beneath appressoria of conidia that had failed to produce secondary hyphae. A pathogenesis-related gene (VvPR-1) was much more highly induced in susceptible berries than in resistant berries after inoculation with U. necator. In contrast, a germin-like protein (VvGLP3) was expressed within 16 h of inoculation in resistant, but not in susceptible berries. Our results suggest that several putative barriers to infection, i.e., cuticle and cell wall thickness, antimicrobial phenolics, and two previously described pathogenesis-related proteins, are not principal causes in halting pathogen ingress on ontogenically resistant berries, but rather that infection is halted by one or more of the following: (i) a preformed physical or biochemical barrier near the cuticle surface, or (ii) the rapid synthesis of an antifungal compound in older berries during the first few hours of the infection process.     

Ultrastructure of the Infection of Sorghum bicolor by Colletotrichum sublineolum

ABSTRACT Ultrastructural studies of the infection of susceptible and resistant cultivars of Sorghum bicolor by Colletotrichum sublineolum were conducted. Initial penetration events were the same on both susceptible and resistant cultivars. Germ tubes originating from germinated conidia formed globose, melanized appressoria, that penetrated host epidermal cells directly. Appressoria did not produce appressorial cones, but each penetration pore was surrounded by an annular wall thickening. Inward deformation of the cuticle and localized changes in staining properties of the host cell wall around the infection peg suggests that penetration involves both mechanical force and enzymic dissolution. In compatible interactions, penetration was followed by formation of biotrophic globular infection vesicles in epidermal cells. Filamentous primary hyphae developed from the vesicles and went on to colonize many other host cells as an intracellular mycelium. Host cells initially survived penetration. The host plasma membrane invaginated around infection vesicles and primary hyphae and was appressed tightly to the fungal cell wall, with no detectable matrix layer at the interface. Necrotrophic secondary hyphae appeared after 66 h and ramified through host tissue both intercellularly and intracellularly, forming hypostromatic acervuli by 114 h. Production of secondary hyphae was accompanied by the appearance of electron-opaque material within infected cells. This was thought to represent the host phytoalexin response. In incompatible interactions, infection vesicles and primary hyphae were formed in epidermal cells by 42 h. However, they were encrusted with electron-opaque material and appeared dead. These observations are discussed in relation to the infection processes of other Colletotrichum spp. and the host phytoalexin response.     

Histopathological Study of Barley Cultivars Resistant and Susceptible to Rhynchosporium secalis

ABSTRACT Differences in the penetration process by Rhynchosporium secalis were compared in resistant and susceptible barley cultivars at the seedling stage. Percent penetration and percent host cell wall alteration (HCWA) differed significantly among cultivars and isolates as revealed by light microscopy. Based on these two variables, the cultivars were statistically separated into two groups that corresponded to their disease reactions. The resistant cultivars, Johnston and CDC Guardian, showed 81.2 to 99.4% HCWA and 0.1 to 20.1% penetration at encounter sites, whereas the susceptible cultivars, Harrington, Argyle, and Manley, had 30.1 to 78.3% HCWA and 31.8 to 81.8% penetration. In the current study, cv. Leduc, which is susceptible at the seedling stage and resistant at the adult stage, showed the same percent HCWA and penetration as did susceptible cultivars. A significant negative correlation (P < 0.01) was found between percent penetration and percent HCWA for cultivars inoculated with two isolates of the pathogen. Isolate 1 was less virulent than isolate 2 with respect to percent penetration and induced significantly fewer HCWA. Scanning electron microscopy showed various shapes of fungal appressoria but no apparent difference in host reaction between resistant and susceptible cultivars. Transmission electron microscopy revealed interactions between the host and pathogen at various stages of penetration. The resistant cv. Johnston responded by producing appositions, as evidenced by a layer of compact osmiophilic material deposited on the inner side of the cell wall. Infection pegs produced by conidia were unable to penetrate the cuticle where an apposition had formed inside. When penetration occurred in the susceptible cv. Argyle, cytoplasmic aggregates and separation of the plasmalemma were visible from the host cell wall, but the layer of compact osmiophilic material was not always present. Data based on light microscopic observations suggested that HCWA may be one of the mechanisms responsible for resistance that is characterized as penetration prevention rather than as a slow rate of mycelial growth after successful penetration. HCWA occurred in response to attempted cuticle penetration, suggesting that HCWA may produce chemical barriers that help to prevent penetration.     

Model Wheat Genotypes as Tools to Uncover Effective Defense Mechanisms Against the Hemibiotrophic Fungus Bipolaris sorokiniana

ABSTRACT We investigated the interaction of several differentially resistant wheatwith the hemibiotrophic phytopathogenic fungus Bipolaris sorokiniana (teleomorph Cochliobolus sativus). Wheat genotypes Yangmai, M 3 (W7976), Shanghai 4, and Chirya 7 showed higher levels of resistancewith cv. Sonalika, used as a susceptible control. In amicroscopic inspection, we found that fungal penetration intoepidermal layer failed mostly through a cell wall-associated defense. In cases where the fungus successfully overcame epidermal, its spread within the mesophyll tissue (necrotrophic phase) wasin the more resistant genotypes. Epidermal cell wall-associated, spreading as well as the extent of electrolyte leakage of infected, correlated well with field resistance. We propose that cellular hostsuch as formation of cell wall appositions as well as the degreeearly mesophyll spreading of fungal hyphae are indicative of thepotential of the respective host genotype and, therefore, could befor the characterization of new spot blotch resistance traits in cereals.     

Pathogen behaviour and plant cell reactions in interactions between Alternaria species and leaves of host and nonhost plants

The growth behaviour of several Alternaria species on leaf discs of host and nonhost plants was examined by u.v. microscopy. Specific patterns of development were found for the pathogens. Differences in development which correlated to host specificity, or which differentiated specialized pathogens from weak or opportunistic pathogens, were obvious only after attempted penetration. Thus, little variation was observed amongst different plants for individual Alternaria species in the rate of germination, in the extent of germ-tube growth or in the frequency of appressorium production. However, plant responses to attempted penetration, which included the formation of callose-containing papillae, callose deposition in the walls of attacked cells and their neighbours and cell necrosis, varied with specific pathogen–plant interactions. Callose deposition occurred at sites of both successful and unsuccessful penetrations and may, therefore, not be a determining factor in the plant–pathogen interactions examined. A biplot technique is used to illustrate the different degrees of host specificity apparent at pre- and post-penetration stages of fungal development.     

小麦与白粉病菌互作中β-1,3-葡聚糖的细胞定位

 利用免疫胶体金方法对小麦与白粉病菌互作过程中β-1,3-葡聚糖酶底物,即β-1,3-葡聚糖的合成与分布情况进行了研究。结果发现,白粉病菌的所有器官包括分生孢子、附着胞、入侵栓、吸器和菌丝中均未发现β-1,3-葡聚糖存在;相反,β-1,3-葡聚糖是小麦各种细胞壁的正常组分,在气孔保卫细胞、纤维细胞、导管分子等的细胞壁中含量非常丰富。病原菌的侵染导致植物细胞中β-1,3-葡聚糖的合成增加,但在乳突结构中不含β-1,3-葡聚糖。以上结果说明,往小麦中导入外源β-1,3-葡聚糖酶基因并高效表达,不会对病原菌结构产生直接的破坏和抑制作用;相反,有时可能会影响到某些植物细胞的正常发育。     

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.     

禾顶囊壳小麦变种对小麦种子根的侵染过程

 光镜和电镜观察表明,禾顶囊壳小麦变种(Gaeumannomyces graminis var.tritici,小麦全蚀病菌)对小麦种子根的侵染过程可分为侵入前、侵入表皮层、进入皮层和进入中柱等4个连续阶段。麦根接菌后在15℃下培养,48 h后侵入表皮层细胞,60 h后进入皮层,120 h后进入中柱。病原菌主要以侵染菌丝直接侵入表皮层,表皮细胞间隙和根毛基细胞是主要侵入部位,少数由附着枝侵入。菌丝穿透细胞壁有明显的酶解作用特征,菌丝先端前方胞壁上还产生电子密物质。皮层细胞是病原菌定殖和发展的主要场所,病原菌还能离解胞间层,形成胞外空间,特别有利于菌丝和菌丝束的扩展。在侵入位点的寄主细胞壁和质膜之间,形成多种形状的木质管,其数量与侵入菌丝的数目相对应,但木质管不能阻止菌丝进入细胞。菌丝进入中柱后,可阻塞导管和筛管。小麦细胞发生退行性病变,尤以细胞壁膨大崩坏和早期质壁分离最明显,细胞间隙还产生性质不明的黄色物质。     

柿树炭疽菌侵染柿树叶柄的超微结构观察

 利用透射电镜技术研究了柿树炭疽菌侵染柿树叶柄的超微结构。结果表明:病原菌侵入寄主细胞后,产生细胞内的初生菌丝,其表面沉积凹凸不平的电子不透明物质。一层界面基质(interfacial matrix)把表初生菌丝细胞壁和凹陷的寄主原生质膜分开。随着初生菌丝定殖下一个细胞,原先细胞中的细胞膜消失,形成许多泡囊,随后叶绿体消失,内质网和高尔基体也逐渐降解,最后细胞内物质全部被降解成电子不透明的颗粒,降解的物质沿着初生菌丝和细胞壁表面沉积。初生菌丝穿透细胞壁的过程中,菌丝顶端接触细胞壁后膨大,并在中部产生一个隔膜,然后顶端细胞产生一个较细的穿透菌丝,穿透寄主细胞壁。穿透菌丝在寄主细胞壁中的狭窄处产生一个隔膜,一旦穿透寄主细胞壁后,迅速膨大。次生菌丝在细胞间和细胞内扩展,通过菌丝体对细胞壁施加的机械压力引起寄主细胞壁破裂,或同初生菌丝一起使细胞壁解体。侵染90 h后,形成垫形分生孢子盘。在分生孢子盘周围的表皮细胞中,次生菌丝不断形成子座组织,使原来的子座扩大,子座不断分化形成产梗细胞,产梗细胞产生分生孢子梗,分生孢子梗生长和发育对角质层和表皮细胞壁组织折叠处施加机械压力,使角质层和表皮细胞壁组织进一步折叠,分生孢子盘也相应扩大。     

细胞乳突的形成和小麦白粉菌成功侵染的关系

 用一套己知抗白粉病单基因的小麦材料,研究了小麦白粉菌诱发寄主表皮细胞产生的乳突与成功侵染的关系。抗病小麦叶片上,诱发产生乳突的分生孢子占测定分生孢子数的58.8%,感病品种为63.8%。这表明抗性不同的小麦材料接种白粉菌之后,叶表皮细胞内形成乳突的百分率相近,然从在抗病材料和感病材料中形成的乳突阻止病原物发育和成功侵染的作用不相同,在抗病叶片上,诱发产生乳突的分生孢于中有88%停留在压力孢阶段,不继续发育,仅有12%能突破乳突形成吸器,使得侵染成功。与此对照,在感病叶片上只有32%诱发乳突的分生孢子不能穿透乳突,而68%则能突破乳突。成功地与寄主建立寄生关系。我们的研究表明,乳突能否成功地阻止白粉菌侵入可能与乳突形成的迟早有关。