Cytological studies on the mode of action of systemic fungicides on the host pathogen complex barley-powdery mildew (Erysiphe graminis f. sp. hordei marchal)

The effects of systemic fungicides on the host-pathogen interface between barley and powdery mildew were studied using cytological methods and specific staining procedures. Propiconazole, etaconazole, bitertanol, triforine, imazalil, nuarimol, tridemorph and fenpropimorph, which are all inhibitors of sterol biosynthesis, stopped growth of the fungus 2 days after application and led to deposits of plant origin around haustoria; these contained polysaccharides, especially callose. This encapsulation resembled the reaction often observed in resistant plants. In contrast, pyrazophos and ethirimol, applied at reduced concentrations, inhibited the fungus at the haustorial stage but did not induce encapsulation. These results suggest that encapsulation is not simply a reaction to the previously damaged fungus, but may be an indirect effect of sterol biosynthesis-inhibiting fungicides on the host metabolism which is elicited by the fungus. The results are discussed in the context of previous observations on the modes of action of these fungicides.     

印度梨形孢的多种功能及其应用前景

印度梨形孢是一种可进行纯培养的类菌根真菌,该菌以菌丝体、菌丝卷、分枝或者圆形体的方式定殖于寄主植物根的胞间和胞内。已有研究表明:该菌能促进12科24种植物的生长,并能诱导植物产生系统抗性和提高对逆境胁迫的忍耐性,加快插枝侧根的形成和促进微繁殖植物的硬化。该菌在根分化区定殖的量高于根尖片断的量,在该菌与植物根部早期的识别过程中,涉及多种蛋白,但这些蛋白与真菌的作用方式并不清楚。另外,该菌在寄主植物上的成功生长涉及到了寄主细胞的死亡,但并未引起植物的逆境,而是在根的生长与真菌的繁殖之间达到了很好的平衡。     

Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. XIV. Evidence for elicitor(s) and suppressor(s) of host inaccessibility from Erysiphe graminis

The release of elicitors and suppressors by Erysiphe graminis, the powdery mildew pathogen of barley, was investigated by microscopy in combination with micromanipulation. The elicitors enhance inaccessibility whereas the suppressors prevent the action of the elicitors. Conidia were deposited onto barley coleoptiles and incubated for intervals that varied from 1-8 h. These conidia were termed the inducer conidia since they determined whether their presence would induce the cells of the host to become inaccessible to subsequent inoculations with the fungus. At specified intervals after inoculation the conidial germlings were removed from cells with a micromanipulator. The coleoptiles were then incubated for an additional 8 h after which, a new germling of the fungus was transferred to the same cell from which the inducer germling had been removed. This new germling was called the challenge germling since it was used to determine if it could challenge the host cell to express either induced accessibility or enhanced inaccessibility. The ability of these challenge germlings to penetrate the barley host cell was then assessed after they had been incubated on the cell for an additional 19 h. This allowed the determination of whether inaccessibility had been enhanced in the host. Inaccessibility was enhanced in the host only when the inducer conidia were incubated on host cells for more than 7 h. Scanning electron microscopy revealed that these challenge germlings did not penetrate into the host cell. Thus, enhanced inaccessibility had occurred. The results indicate that the E. graminis germling released a material that enhanced the inaccessibility of the barley host cells. We refer to this material as an elicitor. The transfer of a challenge germling to a coleoptile was made at various times after the removal of the inducer germling from the tissue. This allowed us to determine that more than 2 h is required for the enhancement of inaccessibility after the removal of the inducer germling from the tissue. If a germling, either the same or a different germling, was left on the host cell continuously, then enhanced inaccessibility did not occur. Rather, susceptibility occurred. These results suggest that the E. graminis germling releases a material that suppresses inaccessibility. We refer to this material as a suppressor. Thus, the results indicate that E. graminis conidia release an elicitor that enhances inaccessibility of barley cells and that they also release a suppressor that prevents enhanced inaccessibility in the barley cell.     

Ultrastructural Studies of the Mode of Penetration by Phoma macdonaldii in Sunflower Seedlings

ABSTRACT An ultrastructural investigation of the artificial inoculation of sunflower with Phoma macdonaldii conidia was undertaken using light, scanning, and transmission electron microscopy to elucidate the host-parasite relationship. The behavior of the conidia deposited on the cotyledon petiole was investigated at various time intervals after inoculation. Conidia adhesion and germination were observed first. The cotyledon petiole was invaded by the fungus directly through the cuticle and via stomata. Externally, the spore and germ tube were covered with a mucilaginous polysaccharide sheath of a cotton-like appearance and of variable thickness. At the time of penetration, the host cuticle was perforated mechanically. The cuticle was slightly depressed and no enzymatic alteration could be observed. The fungus did not form appressoria on the surface of the host tissues but developed an infection peg. As soon as the cuticle barrier was crossed, the fungus rapidly colonized the host parietal layer. In a first step, the plasmalemma of the host cell appeared to be stuck against the cell wall. As soon as the fungus passed through the epidermal cell wall to reach the host cytoplasm, the plasmalemma was disrupted, and the subsequent rapid breakdown of cell integrity favored the colonization of the tissues by the pathogen.     

Stepwise Evolution of Races in Fusarium oxysporum f. sp. ciceris Inferred from Fingerprinting with Repetitive DNA Sequences

ABSTRACT Plant pathogens often exhibit variation in virulence, the ability to cause disease on host plants with specific resistance, evident from the diversity of races observed within pathogen species. The evolution of races in asexual fungal pathogens has been hypothesized to occur in a stepwise fashion, in which mutations to virulence accumulate sequentially in clonal lineages, resulting in races capable of overcoming multiple host plant resistance genes or multiple resistant cultivars. In this study, we demonstrate a simple stepwise pattern of race evolution in Fusarium oxysporum f. sp. ciceris, the fungus that causes Fusarium wilt of chickpeas. The inferred intraspecific phylogeny of races in this fungus, based on DNA fingerprinting with repetitive sequences, shows that each of the eight races forms a monophyletic lineage. By mapping virulence to each differential cultivar (used for defining races) onto the inferred phylogeny, we show that virulence has been acquired in a simple stepwise pattern, with few parallel gains or losses. Such a clear pattern of stepwise evolution of races, to our knowledge, has not been demonstrated previously for other pathogens based on analyses of field populations. We speculate that in other systems the stepwise pattern is obscured by parallel gains or losses of virulence caused by higher mutation rates and selection by widespread deployment of resistant cultivars. Although chickpea cultivars resistant to Fusarium wilt are available, their deployment has not been extensive and the stepwise acquisition of virulence is still clearly evident.     

Anthracnose of Sansevieria trifasciata caused by Colletotrichum sansevieriae sp. nov.

A Colletotrichum sp. was isolated from water-soaked lesions on sansevieria (Sansevieria trifasciata Prain cv. Laurentii) in Japan. Classifying the species only from the morphology of the fungus was difficult; therefore, host range was tested and the ribosomal DNA ITS2 region was phylogenetically analyzed. The fungus was pathogenic only on sansevieria among 20 test plants belonging to 11 families. In a phylogenetic analysis with the neighbor-joining method, the two isolates used formed a single-isolate clade. The fungus is thus proposed to be a new species, Colletotrichum sansevieriae. This report is the first of anthracnose on sansevieria.     

A review of the known unknowns in the early stages of septoria tritici blotch disease of wheat

Septoria tritici blotch (STB) disease of wheat is caused by the fungal pathogen Zymoseptoria tritici. It is the most important foliar disease of wheat in western Europe and affects wheat cultivation worldwide. The combination of intensive fungicide usage, a polycyclic asexual life cycle and an active sexual cycle has led to the emergence of fungal strains resistant/tolerant to all the major classes of fungicides used in its control. The hallmark of this disease is a long, symptomless latent phase that precedes the onset of visible symptoms. Understanding the processes that occur during the symptomless phase of infection is paramount in developing alternative strategies for disease control; however, large gaps in our knowledge of the disease remain. The known unknowns of the latent stage of infection can be summarized in three questions. Does the fungus initiate or manipulate host defences to trigger programmed cell death in order to facilitate nutrient acquisition or is the host acting exclusively? Does the fungus feed during both the latent phase and the necrotrophic phase like a true hemibiotroph? Does the long latent phase serve a beneficial function for the fungus or is it simply an artefact of evolution? This review aims to distil observations made during studies that have directly or indirectly contributed to answering these questions and points towards their most likely answers.     

Purification, Elicitor Activity, and Cell Wall Localization of a Glycoprotein from Phytophthora parasitica var. nicotianae, a Fungal Pathogen of Tobacco

ABSTRACT A glycoprotein of 34 kDa (GP 34) was solubilized at acidic pH from the mycelium of Phytophthora parasitica var. nicotianae and was purified by ion exchange and gel permeation chromatography. Whole tobacco plants treated with GP 34 through their roots showed an enhanced lipoxygenase activity as well as hydroxyproline-rich glycoprotein accumulation, indicating that this molecule had elicitor properties. An antiserum raised against the pure glycoprotein allowed localization of GP 34 by immunogold-labeling on the cell surface of the mycelium when the fungus was grown in vitro. In the wall-less zoospores, GP 34 was limited to the flagellum surface. It was then abundantly synthesized at the onset of encystment. During infection of tobacco plants, labeling was very faint at early stages of colonization, particularly in the susceptible host cultivar. It appeared earlier in the resistant host cultivar and was restricted to the living fungus, declining with mycelium cell death.     

Cytochemical Investigation of the Antagonistic Interaction Between a Microsphaeropsis sp. (Isolate P130A) and Venturia inaequalis

ABSTRACT In an attempt to better understand the mode of action of the antagonistic fungus Microsphaeropsis sp., the interaction between this fungus and Venturia inaequalis was studied, using both light and electron microscopy. Cytological observations indicated that the antagonistic interaction between the two fungi likely involves a sequence of events, including (i) attachment and local penetration of Microsphaeropsis sp. into V. inaequalis hyphae; (ii) induction of host structural response at sites of potential antagonist entry; (iii) alteration of host cytoplasm; and (iv) active multiplication of antagonistic cells in pathogen hyphae, leading to host cell breakdown and release of the antagonist. The interaction was investigated further by gold cytochemistry. The use of gold-complexed beta-1,4-exoglucanase and wheat germ agglutinin/ovomucoid-gold complex to localize cellulosic beta-1,4-glucans and chitin monomers, respectively, resulted in regular labeling of V. inaequalis cell walls. This finding supports other studies refuting the classification of ascomycetes as only a glucan-chitin group. At an advanced state of parasitism, the labeling pattern of cellulose and chitin, which clearly showed that the level of integrity of these compounds was affected, suggested the production of cellulolytic and chitinolytic enzymes by Microsphaeropsis sp. Wall appositions formed in V. inaequalis in response to the antagonist's attack contained both cellulose and chitin. However, penetration of this newly formed material frequently succeeded. This study provides the first detailed picture of the cytological events associated with mycoparasitism in V. inaequalis.     

Cytological and pharmacological evidence that biotrophic fungi trigger different cell death execution processes in host and nonhost cells during the hypersensitive response

Living epidermal cells undergoing a hypersensitive response to penetration by a rust or a powdery mildew fungus exhibited two distinct patterns of cellular changes that occurred from the time of the cessation of cytoplasmic streaming to protoplast collapse. In nonhost cells, this death process was completed in less than 1 h, trans-vacuolar strands initially remained visible after streaming stopped, Brownian-like motion was seen in particulate cytoplasmic components, and the plant nucleus was usually associated with the intracellular fungus. In resistant host cells, trans-vacuolar strands disappeared as cytoplasmic streaming stopped, no motion was seen in the cytoplasm, there was no consistent association of the plant nucleus with the fungus, and the death process took 2 h or more. Neither type of cell death was mimicked by the application of CuCl₂, salicylic acid, or KCN, nor did the individually distinctive morphological features of the cell deaths that they triggered change with chemical concentration. Pharmacological studies suggested that host and nonhost cell death required an influx of extracellular calcium, protein kinase activity and protein synthesis, but not the generation of extracellular reactive oxygen species. The application of peptides with an RGD motif delayed cell death in only one resistant host, and inhibitors of caspase activity delayed cell death only in the nonhost combinations. These and other results suggest that some rust- and powdery mildew-specific resistance genes in host cultivars may control a different cell dismantling process during the hypersensitive response than that triggered in nonhost plants.     

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.     

林芝地区小麦条锈菌转主寄主小檗的鉴定与分布

西藏自治区是我国小麦条锈病发生与流行相对独立的一个区系,小麦条锈病是严重影响西藏小麦安全生产的重要病害之一,林芝地区是西藏自治区小麦条锈病重要的常发区和流行区。已有研究证实,小檗是小麦条锈菌的转主寄主之一,并在我国小麦条锈菌致病性变异产生新菌系和小麦条锈病的发生过程中起作用。林芝地区小檗种类较多,但目前对林芝地区小檗是否能作为小麦条锈菌的转主寄主缺乏相关的研究和报道。本文针对小麦条锈病常发区的西藏林芝地区小檗资源进行了调查,对小檗能否作为小麦条锈菌转主寄主进行了鉴定。结果表明6种小檗在林芝地区常见且分布广泛,均是小麦条锈菌的转主寄主。这对进一步研究西藏小麦条锈病的发生、流行与病原菌致变性变异起着重要作用。     

Simultaneous one-tube quantification of host and pathogen DNA with real-time polymerase chain reaction

ABSTRACT Phaeocryptopus gaeumannii is a widespread foliar parasite of Douglas-fir. Although normally innocuous, the fungus also causes the defoliating disease Swiss needle cast in heavily infected needles. The extent of P. gaeumannii colonization in Douglas-fir foliage was estimated with real-time quantitative polymerase chain reaction (PCR) using TaqMan chemistry. In order to derive a normalized expression of colonization, both pathogen and host DNA were simultaneously amplified but individually detected by species-specific primers and TaqMan probes labeled with different fluorescent dyes. Detection of host DNA additionally provided an endogenous reference that served as both an internal positive control and adjusted for variation introduced by sample-to-sample differences in DNA extraction and PCR efficiencies. The genes employed for designing the TaqMan probes and primers were beta-tubulin for the pathogen and a LEAFY/FLORICAULA-like gene involved in floral development for the tree host. Both probe/primer sets exhibited high precision and reproducibility over a linear range of 4 orders of magnitude. This eliminated the need to analyze samples in multiple dilutions when comparing lightly with heavily infected needles. Quantification of the fungus within needles was successful as early as 1 month after initial infection. Real-time PCR is the only method currently available to quantify P. gaeumannii colonization early in the first year of the colonization process.     

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。     

Cell interactions between a nonpathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain and root tissues of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis>

Nonpathogenic isolates of Fusarium oxysporum can be successful antagonists of pathogenic forms of the same fungal species that commonly attacks crop plants. The characteristics that distinguish nonpathogenic from pathogenic forms are not well understood. In this study, the mode of root colonization of Eucalyptus viminalis seedlings by a nonpathogenic F. oxysporum strain is described at the ultrastructural level. Root systems of E. viminalis plants were inoculated with nonpathogenic F. oxysporum strain Fo47 in an in vitro model system. Changes in the occurrence of nonesterified and methyl-esterified pectins in colonized E. viminalis roots were evaluated by in situ immunolabeling using two monoclonal antibodies, JIM 5 and JIM 7. Modes of penetration and root colonization patterns in E. viminalis seedlings by the nonpathogenic fungus were similar to those described for pathogenic forms of F. oxysporum. However, root interactions differed in that the nonpathogenic fungus did not induce host tissue damage. No papilla-like appositions were observed in host cells in response to invading hyphae, which did not disrupt the host plasma membrane in many cases, suggesting that a biotrophic relationship was established. Root colonization by the nonpathogenic strain did not induce alteration in JIM 7 labeling of methyl-esterified pectin in E. viminalis cell walls, whereas nonesterified pectin was detected to a significantly greater extent in cell walls of roots colonized by the fungus. Pectin components decreased slightly only at points of hyphal contact with host cells. Because nonpathogenic strains utilize pectin in pure culture, host control over enzyme activity or production by the fungi may at least partly explain their compatible interactions with host tissues.     

Virulence factors of Botrytis cinerea

Botrytis cinerea is responsible for gray mold disease in more than 200 host plant species. The infection of host plants is mediated by numerous extracellular enzymes, proteins and metabolites. Each of these compounds may play a role in different stages of the infection process. Cell wall-degrading enzymes may facilitate the penetration into the host surface, while toxins, oxalic acid and reactive oxygen species may contribute to killing of the host cells. Cell wall-degrading enzymes contribute to the conversion of host tissue into fungal biomass. On the other hand, B. cinerea infection induces biosynthesis of phytoalexins. Therefore, the ability to overcome a wide spectrum of phytoalexins contributes to the pathogenicity of the fungus with a broad host range. The cloning of the corresponding genes has facilitated studies on gene expression and targeted mutagenesis. This review gives an overview of the research performed on virulence factors that play the roles in pathogenesis.     

Clonality and long-distance migration of Puccinia striiformis f.sp. tritici in north-west Europe

The biotrophic fungus Puccinia striiformis f.sp. tritici , a basidiomycete that causes yellow rust on wheat, is spread by wind-dispersed spores. Analysis of amplified fragment length polymorphism (AFLP) variation showed that the fungus frequently migrates between the UK, Germany, France and Denmark. There is no biological evidence for sexual or parasexual reproduction under natural conditions, and this was supported by the lack of recombination, as revealed by AFLP, over the time and area represented by the samples in this study. A phylogeographic analysis revealed that there was effectively a single, clonal population in the four countries, up to 1700 km apart, consistent with a 'continent-island' model in which Denmark is the recipient of migrants from other countries. In five cases, specific pathogen clones were dispersed between the UK and Denmark, and on at least two recent occasions clones were also spread from the UK to Germany and France, causing outbreaks of yellow rust on wheat cultivars that were previously resistant to the disease in these countries. The agronomic consequences of migration were enhanced because of the limited genetic diversity for yellow rust resistance in wheat cultivars in the area. These results demonstrate that long-distance migration of pathogen clones, coupled with low diversity in the host species, may cause previously useful resistance genes to become ineffective for disease control on a continental scale.