Substomatal Vesicles Research Articles

Histopathology of Leaf Rust Infection and Development in Wheat Genotypes containing Lr12 and Lr13

Evidence exists that certain genes for resistance to leaf rust in wheat, e.g. Lr13 and Lr34, may interact with other genes to condition higher levels of resistance than that conferred by each gene individually. In this study, the hypothesis that Lr12 and Lr13, both genes for adult plant resistance to Puccinia recondita Roberge ex. Desmaz f. sp. tritici Eriks. and Henn., interact to confer an improved level of resistance, was investigated using fluorescence and phase‐contrast microscopy. Flag leaf segments of monogenic and digenic Thatcher lines, sampled 64 and 240 h post‐inoculation, were stained with Uvitex 2B and screened, using fluorescence microscopy, for development of infection structures or host response. To study cell wall appositions, specimens were stained with trypan blue and a solution of picric acid in methyl salicylate. Aborted penetration, consisting of nonpenetrating appressoria and aborted substomatal vesicles, showed that inhibition of fungal growth in wheat lines containing Lr12 and/or Lr13 was activated, to a certain degree, before haustoria were formed. At 240 h after inoculation colony size indicated that fungal colonies in the Lr gene combination lines were generally smaller than in the parents, but not necessarily smaller than those in a line with Lr13 only. Host cell necrosis was more frequently associated with infection sites, specifically of pathotype UVPrt2, in the combination lines than in the parents. The morphology of cell wall appositions varied considerably from a narrow, luminous zone slightly wider in the centre, to a thick central part opposite the haustorium mother cell, sharply decreasing towards both ends. Histological assessments could, however, not conclusively prove pronounced resistance enhancement or unconventional resistance mechanisms due to combining the genes Lr12 and Lr13.

Disease development and β-1,3-glucanase expression following leaf rust infection in resistant and susceptible near-isogenic wheat seedlings

To determine the association of pathogenesis-related (PR) proteins with resistance conferred by the genes Lr29 and Lr34 to leaf rust, three near-isogenic wheat lines [Palmiet (Pa), Pa/Lr29 (Pa*4/RL6080[Lr29]) and Pa/Lr34 (Pa*4/RL6058 [Lr34])] were infected with the fungus Puccinia recondita f. sp. tritici . Activity levels and an immunological profile of the PR protein, β-1,3-glucanase, were recorded in relation to fungal structure development. Compared with the susceptible control, Palmiet, infection and colonization of leaf tissue were inhibited in lines containing either Lr29 or Lr34 . Induction of β-1,3-glucanase activity was observed in all lines, including Palmiet, concurrent with the formation of substomatal vesicles and subsequent differentiation of the pathogen. Following leaf rust infection, four major β-1,3-glucanases were revealed with Western blot analysis. Higher levels of activity were detected in infected than in non-infected plants. The hypothesis that resistance in lines carrying Lr29 or Lr34 is related to higher levels of β-1,3-glucanase expression was not confirmed.

Microinjected antisense Inf24 oligonucleotides inhibit appressorium development in Uromyces

Germlings of Uromyces appendiculatus respond to topographical stimuli and develop appressoria. During this period several genes are expressed, one of which is Inf 24. Germlings were microinjected with a sense and antisense fragment of the ORF of Inf 24. Sense ( Inf 24-S), 5′-ATG AGT GCT TCA TCG CAG CAG CAG-3′ fragment did not affect germling response to the topographical stimuli and the developed appressoria. Microinjection of antisense ( Inf 24-RC), 5′-CTG CTG CTG CGA TGA AGC ACT CAT-3′ blocked gene transcription and appressoria were rarely formed. Injection of Inf 24-RC into already formed appressoria did not inhibit continued development of subsequent infection structures, e.g. penetration peg, sub-stomatal vesicle.

Scanning electron microscopy of early infection structure formation by Puccinia recondita f. sp. tritici on and in susceptible and resistant wheat lines

The morphology of infection structure development of Puccinia recondita f. sp. tritici on and in susceptible and resistant wheat lines inoculated with urediospores was examined by SEM. The germ-tube extends over the leaf surface and elongates perpendicularly to the long axis of the leaf. When the germ-tube encounters the stomatal lip, an appressorium forms over the stoma and the pore is entered by an infection peg produced on the surface of the appressorium in contact with the host leaf. At 6 h post-inoculation (hpi), infection pegs develop terminally substomatal vesicles (SSVs) in the substomatal chambers of all wheat lines. A septum separates each SSV from its interconnective tube. A primary infection hypha forms terminally from the elongated SSV either parallel to the long axis of the stomatal slit or perpendicular to the leaf surface. When a primary infection hypha attaches to a host cell, a septum forms cutting off the tip of the hypha, delimiting a terminal haustorium mother cell (HMC) by 12 hpi. Secondary infection hyphae arise from a position proximal to, and in the proximity of, the HMC septum. Additional HMCs are formed when a secondary hypha or a tertiary hypha adheres to a plant cell. Infection sites with HMCs were observed at 24 hpi and at subsequent sampling stages. There were no significant differences between the infection processes on the three wheat lines examined in this study.

Development of Early Infection Structures of Puccinia recondita f. sp. tritici in Non‐host Cereal Species

AbstractThe development of infection structures, derived from urediospores of Puccinia recondita f.sp. trilici in nearisogenic lines of susceptible and resistant wheat, and in non‐hosts (viz. maize, oat, sorghum and barley), was examined by fluorescence microscopy and scanning electron microscopy (SEM). The infection structure formation on and in five cereal species follows a similar pattern. In sorghum, fungal development is arrested at the stage of substomatal vesicle formation, while, in maize, most fungal structures collapse during the stage of primary hypha development. By contrast, in wheat, barley and oat, the fungus forms many branched infection hyphae and haustorial mother cells.

Effect of Quantitative Resistance in Wheat on the Development ofPuccinia striiformisDuring Early Stages of Infection

Flag leaves of three quantitatively resistant wheat cultivars and one highly susceptible wheat cultivar were inoculated with urediospores of Puccinia striiformis to study the effect of quantitative resistance on development of the fungus during the first 6 days of the infection cycle. The results indicated that the most important mechanism of resistance is reduction in the frequency of formation of appressoria. On resistant cultivars, twice as many germ tubes failed to produce appressoria and grew over stomata than on the susceptible cultivar. Of the infection structures that oriented themselves on stomata, most were able to form substomatal vesicles. In resistant cultivars, the number of infection sites that developed substomatal vesicles decreased over time. This result suggests a resistance mechanism that disintegrates substomatal vesicles. In addition, the formation of primary infection hyphae is delayed considerably in resistant cultivars. Although large cultivar differences for quantitative resistance have been observed at the cellular level, these cannot fully explain differences at the plant level or field level. Therefore, events that happen after the first 6 days of fungal development, like reduced growth and late abortion, may further explain differences in level of resistance among the quantitatively resistant wheat genotypes.

Mechanisms Associated with Wheat Leaf Rust Resistance Derived fromTriticum monococcum

The infection of wheat (Triticum aestivum) line KS93U9 (Karl*3//PI 266844/PI 355520) with Puccinia recondita f. sp. tritici was studied to determine histological mechanisms of resistance. The development of fungal structures was studied at two host growth stages in KS93U9, its leaf rust-resistant T. monococcum parent lines PI 266844 and PI 355520, and its susceptible recurrent parent 'Karl'. T. monococcum accession Tm2126/5, previously reported to exhibit prehaustorial resistance to P recondita f. sp. tritici, was included in the study. Using fluorescence microscopy, infection sites on leaf sections infected with pathotype UVPrt9 and stained with Uvitex 2B were examined for, respectively, the percentage of prestomatal exclusion of the fungus (germ tubes not forming appressoria and appressoria not forming over stomata), aborted penetration (nonpenetrating appressoria and aborted substomatal vesicles), early abortion (less than six haustorium mother cells per infection site), and successfully established colonies. In general, the resistant lines responded similarly for prestomatal exclusion and aborted penetration, but differences in early abortion and colony formation were observed. In seedlings, prestomatal exclusion could be attributed to the inability of fungal germ tubes to produce appressoria, whereas the formation of nonstomatal appressoria occurred more commonly in adult plants. At both growth stages, most aborted penetration attempts were accounted for by arrested substomatal vesicles rather than nonpenetrating appressoria. All infection sites displaying early abortion in KS93U9 were associated with host cell necrosis, whereas no hypersensitivity was observed in primary leaves of Tm2126/5. In adult Tm2126/5 plants, however, a large proportion of the infection sites exhibited hypersensitivity. Similarly, infection sites in PI 266844 and PI 355520 were frequently accompanied by necrotic leaf tissue. Staining of the leaves with trypan blue and a saturated solution of picric acid in methyl salicylate, and viewing with phase contrast microscopy, showed that papillae commonly occurred at infection sites in the T. monococcum lines, but not in KS93U9. Expression of T. monococcum-derived resistance in KS93U9 was not different from hypersensitive reactions typically associated with existing sources of major gene resistance to wheat leaf rust. Furthermore, components of resistance indicated, that at a histological level, mechanisms of resistance in the hexaploid KS93U9 background were altered when compared with parent lines.

The effects of the fungicide Bayfidan on infection structure formation by Hemileia vastatrix in Coffea arabica cv. Caturra

Treatment of Coffea arabica cv. Caturra seedlings with Bayleton precluded pre-infection structures of the fungus, Hemileia vastatrix, as a target of attack. The fungicide did, however, have an effect on the infection structures within the tissue and fungal disruption occurred between 24 and 48 h post-inoculation (hpi). Extracellular material accumulated on the infection structures. The appearance of the substomatal vesicle and the intercellular hyphae was abnormal, compared to the control, and these structures were swollen. Disruptions in the wall of the infection structures also occurred. Haustorial mother cell collapse was observed 96 hpi, followed by collapse of the entire fungal structure.

The effect of natural and simulated insect herbivory, and leaf age, on the process of infection of Rumex crispus L. and R. obtusifolius L. by Uromyces rumicis (Schum.) Wint.

summaryThe development of uredinia of Uromyces rumicis (Schum.) Wint. was studied on Rumex crispus L. and R. obtusifolius L. plants inoculated in the laboratory. Fewer uredinia developed on leaves injured by simulated insect herbivory, those fed upon by Gastrophysa viridula Degeer (Coleoptera: Chrysomelidae), and young, incompletely expanded leaves, than on uninjured, fully expanded control leaves. The effect of simulated herbivory and leaf age on the process of U. rumicis infection was investigated using Calcofluor staining and epifluorescence microscopy. Simulated insect herbivory reduced significantly the proportion of sporelings producing appressoria and the proportion of sporelings with appressoria that entered the substomatal cavity. In R. obtusifolius, leaf injury also reduced the proportion of penetration hyphae that formed substomatal vesicles. The proportion of sporelings that produced intercellular hyphae in injured leaves was reduced by 58 % in R. crispus and 89 % in R. obtusifolius. G. viridula feeding induced similar resistance in R. obtusifolius leaves.Simulated insect herbivory reduced both uredinial density and the area occupied by intercellular hyphae by between 65% and 89%. Injury had a greater effect than leaf age on sporelings in the pre‐haustorial stages of development. However, the total area occupied by intercellular hyphae in young leaves was reduced by between 42% and 78% compared to mature leaves, owing to a lower uredinial density and, in R. obtusifolius, also to a significant reduction in the size of individual uredinia.

Extracellular Proteases of the Rust Fungus Uromyces viciae-fabae

Rauscher, M., Mendgen, K., and Deising, H. 1995. Extracellular proteases of the rust fungus Uromyces viciae-fabae. Experimental Mycology 19, 26-34. On thigmo-inductive membranes the broad bean rust fungus Uromyces viciae-fabae differentiates complex infection structures including haustorial mother cells. Using this in vitro system, formation of extracellular proteases of the obligately biotrophic fungus was studied during infection structure differentiation. Enzyme activities occur when appressoria are formed, and extracellular washing fluids of substomatal vesicles, infection hyphae, and haustorial mother cells show complex protease patterns on polyacrylamide gels containing gelatin as substrate. The majority of the rust proteases can be classified as metallo-, including Ca 2+-stabilized proteases. The presence of substrate is not required for synthesis of the enzymes. The extracellular proteases, in contrast to intracellular enzymes of this fungus, specifically degrade fibrous, hydroxyproline-rich proteins. Since such proteins are important in plants for cell wall stability and play a role in defense against fungal pathogens, the extracellular proteases of U. viciae-fabae may be involved in localized breaching of the host cell wall.

Development of infection structures by Hemileia vastatrix in resistant and susceptible selections of Coffea and in Phaseolus vulgaris

The sequence of events leading to successful infection of Coffea by Hemileia vastatrix, following the formation of an appressorium over a stoma, was investigated using scanning electron microscopy. In the host, Coffea arabica, a torpedoshaped substomatal vesicle initial develops bilaterally from the apex of the infection wedge, while in the nonhost, Phaseolus vulgaris, the infection wedge protrudes into the substomatal chamber. The substomatal vesicle in both host and nonhost, at 48 h postinoculation, is anchor shaped. Haustorial mother cells are formed on stubby primary infection hyphae that curve back onto subsidiary cells. No differences in appearance of these structures were noted between resistant and susceptible coffee selections. A much-branched mycelium ramifies through the intercellular spaces of the mesophyll cells 96 h postinoculation in the host. In bean, the SSV began to collapse 48 h postinoculation. Key words: coffee leaf rust, infection, penetration, Coffea, appressorium, substomatal vesicle.

The infection of white clover ( Trifolium repens) by conidia of Cymadothea trifolii

Infection of white clover by conidia of Cymadothea trifolii was through stomata on the adaxial leaf surface only. Spores failed to germinate on the abaxial surface, possibly due to a difference in the structure or composition of leaf waxes. Germ-tubes showed preferential growth along the epidermal wall junctures. A high relative humidity, between 98 and 100%, was necessary for germination. Light did not impair infection. High temperature increased the percentage germination and the number of germlings forming appressoria. More appressoria formed sub-stomatal vesicles at 15 °C than at 8 and 22°.

Characterization and partial purification of pectinesterase, a differentiation-specific enzyme of Uromyces viciae-fabae

The differentiation-specific formation of three isoforms of pectinesterase by the broad bean rust fungus Uromyces viciae-fabae is described. Activity becomes detectable when substomatal vesicles are formed. In crude extracts isoform A contributed 78% of the total pectinesterase activity, and isoforms B and C contributed 20% and 2%, respectively. All three isoforms were found extracellularly in ratios identical to those in extracts. The isoelectric points of the pectinesterase isoforms were 8.4 (A), 5.7 (B), and 4.7 (C) as determined by chromatofocusing. Isoform A had an M r of 33500 and showed a distinct pH optimum at 6.0. The M r of isoform B was 40000; its pH optimum ranged from 5.5 to 7.5. Due to its extremely low activity, isoform C was not further characterized. The possible role of pectinesterases in the infection process of the broad bean rust fungus is discussed.

Carbohydrates on the surface of urediniospore‐ and basidiospore‐derived infection structures of heteroecious and autoecious rust fungi

summaryNine flucirescein isothiocyanate‐labelled lectins with affinity towards different carbohydrates were used to probe the surface carbohydrates of infection structures in vitro, derived from urediniospores and basidiospores of an autoecious rust species, Uromyces viciae‐fabae (Pers. J Schroet., and of a heteroecious rust species, Uromyces rumicis (Schum.) Wint. Lectin binding was quantified by measuring fluorescence photometrically.All lectins bound in a characteristic pattern to the infection structures of the respective spore types of each rust fungus. Differences were especially obvious between those infection structures normally produced inside leaf tissue, namely the urediniospore‐derived substomatal vesicles with infection hyphae, and the basidiospore‐derived intraepidermal vesicles. The dikaryotic stage and the monokaryotic stage of the heteroecious fungus differed mainly in their affinity for the leotins from Bandeira simplicifolin and Lotus tetragonulobus.A statistical analysis comparing the binding of the lectins to infection structures of Two rust fungi suggested that cell surface composition is determined by nuclear condition. The monokaryotic stages of both rust fungi have a higher degree of similarity than the dikaryotic and monokaryotic stages of the same rust fungus.

Infection of and Colony Development within Leaves of Susceptible and Resistant Pearl Millet and Two Nonhosts by the Rust Fungus Puccinia Substriata Var. Indica

The rust fungus Puccinia substriata var. indica produced germ tubes, appressoria, and substomatal vesicles on seedling and mature leaves of susceptible and resistant pearl millet as well as two nonhost plants, corn and peanut. Lack of directional germ tube growth on peanut leaves appeared to affect the ability of germ tubes to successfully locate stomates. In seedling leaves of the susceptible cultivar Tift 23DB, the fungus produced an extensive system of intercellular hyphae and sporulated abundantly. In seedling leaves of the moderately resistant cultivar 86–8770, necrosis of host cells in response to fungal infection occurred by 2 days post-inoculation. Colonies gradually expanded in size and were associated with macroscopic necrotic flecks with sporulation occurring at 15% of the infection sites. In seedling leaves of the highly resistant cultivar Tift 85DB, fungal colonies were extremely limited in growth and did not sporulate. They were associated with intensely autofluorescent, necrotic host cells. Mature leaves of each cultivar were more resistant to fungal colonization than seedling leaves. Some colonies in mature leaves of Tifit 23DB were completely encompassed by necrotic cells and did not expand further. No sporulation was noted in mature leaves of 86–8770 or Tift 85DB. Fungal colonies were not successfully established in either nonhost. Thickening of plant cell walls at sites of attempted infection was noted in corn. In peanut, guard cells underneath fungal appressoria became autofluorescent.

Surface carbohydrates and cell wall structure of in vitro-induced uredospore infection structures ofUromyces viciae-fabae before and after treatment with enzymes and alkali

Uredospores ofUromyces viciae-fabae differentiate to form germ tubes, appressoria, infection hyphae and haustorial mother cells on oil-containing collodion membranes. The cell walls of these infection structures were studied with the electron microscope and with FITC-labeled lectins before and after treatment with enzymes and inorganic solvents. Binding of the FITC-labeled lectins was measured with a microscope photometer. The enzymes pronase E, laminarinase, chitinase and lipase had different effects on each infection structure. Pronase treatment uncovered the chitin of germ tubes, appressoria and haustorial mother cells, but not of substomatal vesicles and infection hyphae. A mixture of α- and β-1,3-glucanase which also contained chitinase activity dissolved germ tubes and appressoria completely, but not infection pegs, substomatal vesicles, infection hyphae and haustorial mother cells. After treatment with laminarinase or lipase, an additional layer, which is especially obvious over the substomatal vesicle, infection hypha and haustorial mother cell, bound to LCA-FITC. In the wall of the haustorial mother cell, a ring, which surrounds the presumed infection peg, had strong affinity for WGA after protease and sodium hydroxide treatment. The infection structures have a fibrillar skeleton. The main constituent seems to be chitin. This skeleton is more dense or has a higher chitin content in the walls of appressoria and haustorial mother cells. The fibrils of the skeleton extend throughout the cell wall of the germ tube and appressorium. They are embedded within amorphous material of complex chemical composition (α-1,3-glucan, β-1,3-glucan, glycoprotein). The chitin of the infection peg, substomatal vesicle, infection hypha and haustorial mother cell is covered completely with this amorphous material. These results show, that each infection structure has distinct surface and wall characteristics. They may reflect the different tasks of the infection structures during host recognition and leaf penetration.

Temporal and spatial dynamics of appressorium formation in Uromyces appendiculatus

Appressorium development in Uromyces appendiculatus was studied to establish temporal and spatial parameters possibly important in signaling mechanisms in this fungus. It was determined that urediospore germling apices sensed inductive topographies, e.g., precisely shaped ridges 0.5 μm high by 2.0 μm wide, within 4 minutes after initial contact. The area in the germling that senses such topographies was located on the substrate side of the cell and within 6.0 μm of the apex. These conclusions were based on observed changes in the rate of germling growth and nuclear migration as well as the timing of apical tip swelling that began 4 minutes after contact. DNA synthesis during appressorium development occurred only after the first nuclear division and was probably in preparation for the second mitosis that normally occurs in the substomatal vesicle. Treatments with the DNA synthesis inhibitors hydroxyurea and actinomycin D reduced the DNA content of individual nuclei only after nuclear division. These results indicate that the nuclei in urediospore germlings were already in the G2 phase when germination began. Formation of a septum during appressorium development was initiated 45–50 minutes after contact of the germling with the ridge and was completed within 60 minutes after all of the cytoplasm had entered into the swollen apex.

Differentiation-related proteins of the broad bean rust fungus Uromyces viciae-fabae, as revealed by high resolution two-dimensional polyacrylamide gel electrophoresis

On artificial polyethylene membranes providing a thigmotropic signal, uredospores of the broad bean rust fungus Uromyces viciae-fabae differentiated a series of infection structures which in nature are necessary to invade the host tissue through the stomata. Within 24 h germ tubes, appressoria, substomatal vesicles, infection hyphae and haustorial mother cells were developed successively. Alterations in protein metabolism during infection structure differentiation of this obligate plant pathogen were analyzed in the absence of the host plant by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver staining. The norm pattern representing the 2-DE protein patterns of the whole developmental sequence of infection structures of U. viciae-fabae showed 733 spots. During infection structure differentiation 55 proteins were newly formed, altered in quantity, or disappeared. Major alterations in the protein pattern occurred during uredospore germination and when infection hyphae were formed. Uredospore germination was characterized by a decrease of acidic proteins and an increase mainly of proteins with isoelectric points ranging from weakly acidic to basic.

Ultrastructural Morphology ofUromyces transversalisInfection of Resistant and Susceptible Gladiolus Hosts and a Nonhost,Zea mays

The infection structures of gladiolus rust, Uromyces transversalis, on and in host leaves of the susceptible gladiolus cultivar (Goldfield) and resistant species (Gladiolus daleni) and in leaves of the nonhost (Zea mays) were examined by scanning and transmission electron microscopy. The number of germinated urediospores that formed appressoria on the resistant host was significantly fewer than the number formed on the susceptible host. The major determinant of resistance in the host was manifested in the significant proportion of substomatal vesicles with primary hyphae that aborted before the formation of haustorial mother cells and/or secondary hyphae (...)

Directional growth of wheat leaf rust infection structures after penetration and formation of the substomatal vesicle

Wheat leaf rust infection structures showed preferential growth towards the leaf vein. There was no indication that this directional growth was induced by morphological features of the leaf tissue