The evolutionary significance of variation in infection behavior in two species of rust fungi on their hosts and related nonhost plant species

We explored the association between the phylogeny of 14 species of beans in the genera Phaseolus and Vigna (subtribe Phaseolinae) and the progress of infections in them caused by two pathogenic rust fungi, Uromyces appendiculatus (Pers.) Unger and Uromyces vignae Barclay. The analyses involved construction of phenograms, ordinations, and cladograms derived from the infection characteristics and their comparison to a composite of three previously published DNA phylogenies. The patterns of infection by U. vignae showed no relationship to the phylogeny of the infected plants. The course of infection by U. appendiculatus, however, displayed a significant phylogenetic component, although the defense responses of the infected plants themselves did not. These differences in phylogenetic signal between infections caused by the two fungi may reflect distinct evolutionary histories with members of the Phaseolinae, a long association for U. appendiculatus but a much more recent one for the more host-specific U. vignae.Key words: bean rust, cowpea rust, evolution, infection characteristics, Phaseolus, Vigna.

Changes in the plant endomembrane system associated with callose synthesis during the interaction between cowpea (Vigna unguiculata) and the cowpea rust fungus (Uromyces vignae)

Electron microscopy and stereological analysis of cowpea (Vigna unguiculata) leaf tissue infected with the cowpea rust fungus (Uromyces vignae) revealed an increase in surface of plant endomembranes that was associated with callose synthesis or the presence of fungal haustoria. In the resistant cultivar in which the haustorium commonly becomes encased, an increase in surface of smooth membranes was observed in cytoplasmic regions adjacent to developing encasements compared with the regions away from the fungus or with any region in infected or uninfected callose nonsynthesizing cells. Cytoplasmic regions adjacent to the haustorium in callose nonsynthesizing cells had an increase in rough endoplasmic reticulum (ER). This increase was greater in a susceptible cultivar than in the resistant cultivar that was treated with tunicamycin to inhibit callose synthesis. In the latter situation, the lack of callose encasement allowed the haustorial neckband to form, but other ultrastructural signs of incompatibility remained, such as the presence of electron-opaque material associated with the extrahaustorial membrane. No differences between cultivars or treatments were observed in Golgi membranes. Our observations suggest that both callose synthesis and fungal presence are associated with de novo synthesis of membranes; callose deposition may require an increase in smooth membranes of uncertain origin, whereas the establishment of a haustorium may be dependent on increased synthesis of rough ER. Key words: callose, endoplasmic reticulum, resistance, stereology, tunicamycin.

Genetics and cytology of age-related resistance in North American cultivars of cowpea (Vigna unguiculata) to the cowpea rust fungus (Uromyces vignae)

Increasing leaf age was accompanied by increases in resistance in three incompatible cowpea cultivars inoculated with race 1 of Uromyces vignae and in three of four cultivars that had previously been considered susceptible to race N2. Selected crosses between cultivars suggested that resistance to race 1 was controlled by the same genes in young and old leaves, and they indicated that age-related resistance to race N2 was primarily controlled by a single, and different, gene in each cultivar. All examples of resistance were expressed cytologically as a range of infection site types in a single leaf, the frequency distribution of which was affected by the type of resistance gene, gene heterozygosity, and leaf age. These frequency distributions shifted with increasing leaf age towards infection sites with less fungal growth and more rapid plant cell necrosis, often abolishing the cytological differences between cultivars and genotypes seen in younger plants. The data suggest that each rust resistance gene in cowpea can generate a range of infection site types, and that fungal growth and plant responses at each infection site are governed by a combination of the number and type of resistance genes in the plant, the race of the fungus, and age-affected features of individual plant cells. Key words: cowpea, rust fungi, age-related resistance.

Differentiation of aecidiospore- and uredospore-derived infection structures on cowpea leaves and on artificial surfaces by Uromyces vignae

Aecidiospores and uredospores are the two dikaryotic spore forms of the cowpea rust fungus Uromyces vignae. After germination they can be induced to develop a series of infection structures including appressoria, infection hyphae, and haustorial mother cells. Haustoria are then formed within host cells. The differentiation of infection structures was compared on polystyrene membranes with defined topographies, on scratched polyethylene membranes, and in planta. On polystyrene membranes with defined topographies both sporelings showed highest rates of differentiation on ridges 0.3 μm high but aecidiosporelings responded less efficiently to this stimulus than uredosporelings. On scratched polyethylene membranes, almost 90% of both sporelings differentiated appressoria, but only 10% formed haustorial mother cells; haustoria were not observed. On the host plant, by contrast, only 50% of the sporelings differentiated appressoria, but most of these formed haustorial mother cells and haustoria. In planta haustorial mother cell development occurred approximately 6 h earlier than on inductive membranes. Infection structures formed on artificial membranes and on host plants were similar in morphology and nuclear condition. Key words: cowpea rust fungus, nucleus, appressorium.

Early events in living epidermal cells of cowpea and broad bean during infection with basidiospores of the cowpea rust fungus

The infection process of basidiospores of the cowpea rust fungus (Uromyces vignae) was studied on living host (Vigna sinensis) and nonhost (Vicia faba) leaves using light microscopy with differential interference contrast optics. During the first 8 h, fungal development was similar on host and on nonhost leaves. Penetration and production of intraepidermal vesicles occurred in nonhost cells 4 h earlier than in host cells. Penetration frequency was also higher in nonhost leaves. These results suggest that the cuticle of the cowpea plant delays basidiospore infection. Both host and nonhost cells produced cytoplasmic aggregates during appressorium formation and again, a few hours later, during penetration of the epidermal cell wall. Cytoplasmic aggregates were also observed in cells adjacent to a collapsing cell. Papillae were observed at most infection sites in both host and nonhost cells. The nuclei of infected cells migrated towards the penetration site in both plant–pathogen combinations. Nuclear size increased considerably only in the nonhost epidermis and decreased again markedly after cell collapse. In the nonhost cells, three types of defence reactions occurred during or after formation of the intraepidermal vesicle. First, following the halt of cytoplasmic streaming, the cytoplasm of the invaded cell either contracted or disintegrated into granules. Alternatively, the cytoplasm continued streaming but darkly pigmented material encased the fungal infection structure. Key words: basidiospore, broad bean (Vicia faba), cowpea (Vigna sinensis), cowpea rust fungus (Uromyces vignae), hypersensitivity, nonhost resistance.

Cytological studies of the hypersensitive death of cowpea epidermal cells induced by basidiospore-derived infection by the cowpea rust fungus

The cytological responses to the monokaryotic primary hyphae of the cowpea rust fungus (Uromyces vignae Barcl.) were observed in vein epidermal cells of a resistant and a susceptible cowpea cultivar. Unlike the previously examined response to haustoria of a nonpathogenic powdery mildew fungus, plant cell walls did not become autofluorescent in response to fungal penetration, and the primary hypha only rarely became encased. Following fungal penetration, the response of invaded cells of the resistant, intact plant could be divided into the following stages: (I) cytoplasmic streaming normal; (II) cytoplasmic streaming slow or stopped, Brownian motion of particles visible in the vacuole, granulated cytoplasm aggregated along the cell walls, some host nuclei disappeared; and (III) protoplast collapsed. Epidermal tissue of the resistant cultivar did not exhibit stages II–III when detached and mounted in water 12 h after inoculation and examined 9 h later. The frequency of stage III increased when the tissue was mounted in CaCl2, Ca(NO3)2, and KNO3, but only in a kinetin solution did it approximate that in attached tissue. Although kinetin inhibited fungal growth in both the resistant and the susceptible cultivar, the hypersensitive response occurred only in the former, suggesting that kinetin affects the hypersensitive response directly rather than through its inhibitory effect on the fungus. Key words: cowpea, Vigna unguiculata, cowpea rust fungus, Uromyces vignae (Barcl.), hypersensitivity.

In vitro formation of haustoria of the cowpea rust fungus Uromyces vignae in the absence of a living plant cell. II. Electron microscopy

The ultrastructure of infection hyphae, haustorial mother cells, and haustoria of Uromyces vignae formed on collodion membranes is described after conventional preparation and after treatment with periodic acid – thiocarbonhydrazide – silver proteinate or periodic acid – chromic acid – phosphotungstic acid treatments. Infection hyphae and haustorial mother cells developed normally in vitro but lacked an outer wall layer present in older infections in the host plant. Haustorium formation in vitro was accompanied by the development of elaborations of the plasmalemma along the infection hypha side of the haustorial mother cell septum, but their contents did not show identical staining responses to the haustorial neck wall as reported for other rust fungus species. Haustorial necks, and to a lesser extent haustorial bodies, were coated with a fibrillar material, the staining characteristics of which were similar to material normally considered part of the extrahaustorial matrix in infected plants. The restriction of this material to the haustorium suggests that it may play an important role in the interaction between the haustorium and the plant cell.