An Investigation into the Involvement of Defense Signaling Pathways in Components of the Nonhost Resistance of Arabidopsis thaliana to Rust Fungi Also Reveals a Model System for Studying Rust Fungal Compatibility

Seventeen accessions of Arabidopsis thaliana inoculated with the cowpea rust fungus Uromyces vignae exhibited a variety of expressions of nonhost resistance, although infection hypha growth typically ceased before the formation of the first haustorium, except in Ws-0. Compared with wild-type plants, there was no increased fungal growth in ndr1 or eds1 mutants defective in two of the signal cascades regulated by the major class of Arabidopsis host resistance genes. However, in the Col-0 background, infection hyphae of U. vignae and two other rust fungi were longer in sid2 mutants defective in an enzyme that synthesizes salicylic acid (SA), in npr1 mutants deficient in a regulator of the expression of SA-dependent pathogenesis related (PR) genes, and in NahG plants containing a bacterial salicylate hydroxylase. Infection hyphae of U. vignae and U. appendiculatus but not of Puccinia helianthi were also longer in jar1 mutants, which are defective in the jasmonic acid defense signaling pathway. Nevertheless, haustorium formation increased only for the Uromyces spp. and only in sid2 mutants or NahG plants. Rather than the hypersensitive cell death that usually accompanies haustorium formation in nonhost plants, Arabidopsis typically encased haustoria in calloselike material. Growing fungal colonies of both Uromyces spp., indicative of a successful biotrophic relationship between plant and fungus, formed in NahG plants, but only U. vignae formed growing colonies in the sid2 mutants and cycloheximide-treated wild-type plants. Growing colonies did not develop in NahG tobacco or tomato plants. These data suggest that nonhost resistance of Arabidopsis to rust fungi primarily involves the restriction of infection hypha growth as a result of defense gene expression. However, there is a subsequent involvement of SA but not SA-dependent PR genes in preventing the Uromyces spp. from forming the first haustorium and establishing a sufficient biotrophic relationship to support further fungal growth. The U. vignae-Arabidopsis combination could allow the application of the powerful genetic capabilities of this model plant to the study of compatibility as well as nonhost resistance to rust fungi.

Host – parasite relationships between the fungus Leptosphaerulina crassiasca and peanut

The mode of penetration and infection of the peanut leaf by Leptosphaerulina crassiasca were studied by means of light and electron microscopy. The attachment of the multicellular ascospores to the leaf surface was by a mucilagenous sheath that covered the ascospores at maturity. This sheath expanded rapidly in moisture and it extended along the germ tube as it elongated. Two types of germ tubes appeared to be formed, a short one and a relatively long one. Short germ tubes were not delimited by septa, and they penetrated the cuticle and host epidermal cell wall directly without appressorium formation. Penetration occurred 2–6 h after inoculation. The wall was breached by a relatively broad infection hypha that expanded in width inside the host cell wall. The lack of mechanical rupture at the infection site indicated that penetration may involve enzymatic activity. Intracellular hyphae were present in the epidermal cells, but only intercellular hyphae occurred in the palisade and spongy mesophyll tissues. The intercellular hyphae were frequently appressed to the outer surface of the host cell wall. Infected areas rarely exceeded 1 mm in diameter, and they were only sparsely colonized by hyphae of the pathogen. Host cells in the vicinity of hyphae underwent senescence and death. One to 2 months after inoculation, pseudothecia formed in the dead tissues of detached leaves. In some instances the presence of penetration hyphae by short germ tubes induced the formation of a papilla inside the host cell wall, which either restricted growth of the infection hypha or resulted in the death of the germ tube and the cell from which it arose. Long germ tubes were delimited by simple septa and they terminated in an appressorium; however, details of their behavior were not studied. Key words: Arachis hypogaea, Ascomycotina, Dothideales, leaf scorch, pepper spot.

Interactions of two strains of Magnaporthe grisea with rice, goosegrass, and weeping lovegrass

Two strains of the fungus Magnaporthe grisea caused responses in goosegrass, weeping lovegrass, and rice that varied from no visible symptoms, through isolated discrete lesions with or without brown margins, to coalescing lesions that completely killed the inoculated areas of the leaf. The fungus sporulated under conducive conditions in all of the five plant–strain combinations that produced visible symptoms, but the degree of sporulation varied considerably. Microscopical examination revealed a previously unreported dimorphism in the infection hypha in all three plant species. In all combinations, some appressoria failed to form infection hyphae. In all but one combination, some infection hyphae failed to differentiate and (or) became restricted to a single epidermal cell with autofluorescent walls. Quantitative data showed that the presence and density of lesions reflected the frequency with which appressoria formed growing fungal colonies; lesion appearance depended on the interaction between such colonies and the leaf tissue. The results suggest that the different plant–strain combinations represent a continuum of plant–fungus interactions, and that small differences in the frequency of different plant–fungus interactions at the cellular level may have significant effects on macroscopic symptom development.

Ultrastructure of the early stages of infection of peanut leaves by the rust fungus Puccinia arachidis

Peanut rust disease proved to be an excellent system for ultrastructural study of development of infection structures by the fungus Puccinia arachidis. Fungal structures were clearly visible by light microscopy in fixed and embedded samples and could be located either on leaf surfaces or within the large substomatal chambers of peanut leaves. Samples could easily be oriented for thin sectioning. The infection process was a highly orchestrated process involving precisely timed events and highly specialized structures. Infection pegs developed from appressoria over stomata and entered the leaf by growing into the openings between guard cells. Once past the rim formed by the guard cell walls, the infection peg expanded to form a substomatal vesicle in which a synchronous mitotic division of the four nuclei occurred. A primary infection hypha then developed from the vesicle and grew into the mesophyll of the leaf until its tip or side contacted a host cell. A septum then delimited a binucleate or trinucleate terminal haustorial mother cell from the remainder of the infection hypha. The wall of the haustorial mother cell became closely appressed to that of the host cell. Following differentiation of the haustorial mother cell, a penetration peg arose from it and penetrated the host cell wall. The peg invaginated the host cell plasma membrane as it elongated and then expanded at its tip to form the haustorium body into which most of the contents of the haustorial mother cell moved. Meanwhile, the primary infection hypha formed secondary hyphae that gave rise to additional haustorial mother cells and haustoria. Key words: Puccinia arachidis, peanut rust, infection process, ultrastructure.

Ultrastructure of conidial germ-tube development in vitro by the insect pathogen Entomophaga aulicae

In an effort to understand the factors influencing the formation of infection structures by the insect pathogen Entomophaga aulicae, we examined the ultrastructural development of conidial germ tubes formed in vitro under conditions that resemble those producing appressoria and protoplasts during infection. Conidia germinated on formvar in a nonaqueous environment produced a single viable germ tube, which in turn produced appressorium-like structures or secondary conidia, structures similar to those formed on the host. The formation of appressorium-like structures indicates that stimuli for appressorium formation are relatively nonspecific, whereas development of the infection hypha requires different triggers. Conidia germinated in a liquid culture medium, which supports the growth of the protoplast stage of the fungus, produced a single viable germ tube that continued to grow apically. This apical growth was accompanied by the loss of the outer layer of the germ tube wall and the presence of electron-opaque granules in an extensive system of cytoplasmic membranes. Protoplasts did not form directly from these germ tubes and ultrastructural details of tip growth in this medium did not resemble those previously described in infection hyphae prior to protoplast formation in the host.

Haustorial mother cell development by Uromyces vignae on collodion membranes

The development of infection structures by the rust fungus Uromyces vignae was observed on oil-containing collodion membranes. About 40% of infection hyphae formed a haustorial mother cell, but this structure commonly senesced and died more rapidly than the infection hypha to which it was attached. These data suggest that the continued development of the haustorial mother cell requires some component normally provided by the host plant. Before they died, many haustorial mother cells apparently formed the thickened region of the wall which normally is traversed by the penetration peg during haustorium formation. Such a peg was observed in the centre of up to 40% of these thickened regions. However, no pegs protruded beyond the haustorial mother cell far enough to be called a haustorial neck. The thickened region of the haustorial mother cell wall could be differentiated from the rest of the wall by its lack of fluorescence under ultraviolet irradiation when mounted in Calcofluor or SITS (4-acetomido-4′-iso-thiocyanatostilbene-2,2′-disulphonic acid). Treatment with alkali, acid, chloroform–methanol, protease, and laminarinase did not affect this differential fluorescence, and the haustorial mother cell wall stained uniformly for proteins, carbohydrates, and chitin. Since Calcofluor normally binds to chitin, these data suggest that the thickened region of the haustorial mother cell wall may physically exclude the dye or may contain potential binding sites that are masked by other wall components.

Cellular differentiation in Venturia inaequalis ascospores during germination and penetration of apple leaves

The penetration of the apple leaf cuticle by ascospores of Venturia inaequalis was studied by using ultrastructural and cytochemical techniques. A time-course developmental sequence is presented. Attachment of ascospores to the leaf surface appears to be by a mucilaginous substance and is followed by germination and penetration pore formation. The penetration pore is a circular opening adjacent to the leaf. It appears to form by degradation of the fungal wall and is bordered by a thickening of fungal wall material. Above the penetration pore, an infection sac forms from invagination and extension of the fungal plasmalemma. This structure has been reported only in V. inaequalis. Infection sac development initiates when the fungal plasmalemma appears to be forced into a dome shape. The dome flattens out and the membrane folds back upon itself, circumscribing the margin of the pore. The folded membrane becomes apposed, resulting in a circular belt of junctional structures which morphologically resemble molluscan septate junctions. As the infection sac enlarges, additional junctions form wherever the infection sac membrane abuts with the fungal plasmalemma. Selective staining with phosphotungstic acid revealed that the infection sac membrane becomes differentiated from the fungal plasmalemma. The infection sac enlarges and accumulates a dense matrix that appears to penetrate into the cuticle, causing ultrastructural changes in host tissues. The infection hypha, which is an extension of the infection sac membrane, breaches the cuticle without any apparent mechanical pressure.

Ultrastructure of the infection of spruce budworm larvae by the fungus Entomophaga aulicae

The infection of spruce budworm larvae by the fungus Entomophaga aulicae was studied by light, scanning, and transmission electron microscopy. Exposure of the larvae to conidia resulted in the death of the host and the production of a crop of infective conidia within 5 to 6 days. Germinating conidia produced appressoria prior to penetration of the larval cuticle. Nuclei in appressoria were larger and contained less heavily condensed chromatin than nuclei in other stages of fungal development. Attachment and infection were characterized by distinct changes in the wall layers of the fungus. An infection hypha grew through the insect tissues directly into the hemocoel where discontinuities were observed in the wall layers at the apex of the infection hypha. These discontinuities are probably associated with protoplast formation in vivo. Protoplasts with polysaccharide and lipid storage material were present in the hemolymph of larvae 3 days after exposure to conidia. Thick-walled hyphal bodies with abundant lipid reserve filled the larval body cavity by the 5th day. Hyphal bodies germinated to produce unbranched, aseptate conidiophores which emerged through the host cuticle prior to conidiogenesis.